Transcriptional repression leading to parkinson&#39;s disease

ABSTRACT

Parkinson&#39;s disease is caused by the preferential loss of substantia nigra dopamine neurons. A Parkin Interacting Substrate, PARIS (ZNF746) is identified. The levels of PARIS are regulated by the ubiquitin proteasome system via binding to and ubiquitination by the E3 ubiquitin ligase, parkin. PARIS is a KRAB and zinc finger protein that accumulates in models of parkin inactivation and in human brain Parkinson&#39;s disease patients. PARIS represses the expression of the transcriptional co-activator, PGC-1α and the PGC-1α target gene, NRF-1 by binding to insulin response sequences in the PGC-1α promoter. Conditional knockout of parkin in adult animals leads to progressive loss of dopamine (DA) neurons that is PARIS dependent. Overexpression of PARIS causes selective loss of DA neurons in the substantia nigra, which is reversed by either parkin or PGC-1α co-expression. The identification of PARIS provides a molecular mechanism for neurodegeneration due to parkin inactivation.

RELATED APPLICATIONS

This is application is a Continuation of U.S. Non-provisionalapplication Ser. No. 14/056,409 filed on Oct. 17, 2013, which is aContinuation of U.S. Non-provisional application Ser. No. 13/294,909filed on Nov. 11, 2011, now U.S. Pat. No. 8,603,994, issued on Dec. 10,2013, and which claims priority to U.S. Provisional Application No.61/412,426 filed on Nov. 11, 2010, the content of which is hereinincorporated by this reference in its entirety. All publications,patents, patent applications, databases and other references cited inthis application, all related applications referenced herein, and allreferences cited therein, are incorporated by reference in theirentirety as if restated here in full and as if each individualpublication, patent, patent application, database or other referencewere specifically and individually indicated to be incorporated byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant awardnumbers NS38377, NS048206 and NS051764 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates the detection, diagnosis, and treatment ofParkinson's disease and related disorders. More particularly, thepresent invention relates to isolated polypeptides, isolatedpolynucleotides, compositions, methods and kits for the detection,diagnosis, and treatment of Parkinson's disease and related disorders.Additionally, the present invention relates to the biochemical factorsand biochemistry of the causative agents of Parkinson's disease andrelated disorders.

BACKGROUND OF THE INVENTION

Parkinson's disease (PD) is a progressive neurodegenerative disorderthat is characterized phenotypically by bradykinesia, rigidity, tremor,and neuropsychiatric disturbances (Savitt et al., 2006). Although thecause of PD in the majority of cases is unknown, there are rare familialcases for which the genes have been identified. There are at leastsixteen PD associated loci (Gasser, 2007). Mutations in α-synuclein andleucine rich repeat kinase 2 (LRRK2) cause autosomal dominant PD. Fourgenes have been linked to autosomal recessive PD (AR-PD) and includemutations in parkin, DJ-1, PINK1, and ATP13A2. Investigating the biologyof these genes and their mutant protein has provided tremendous insightinto the pathogenesis of both familial and sporadic PD (Gasser, 2007;Savitt et al., 2006).

Parkin is an ubiquitin E3 ligase (Shimura et al., 2000; Zhang et al.,2000). In general, PD-associated mutations in parkin lead to loss of itsE3 ligase function (Tanaka et al., 2004). Moreover, oxidative,nitrosative, and dopaminergic stress, which play important pathogenicroles in PD, inactivate parkin, suggesting that parkin inactivation mayplay a role in sporadic PD (Chung et al., 2004; LaVoie et al., 2005;Winklhofer et al., 2003). Thus, substrates of parkin that are subject toproteasomal degradation should accumulate in animal and cellular modelsof parkin inactivation and AR-PD due to parkin mutations, and also insporadic PD. There are a diverse array of parkin substrates that hashindered the generation of a consensus in the field on parkin'sphysiologic function and pathologic role in PD. Moreover, parkin'sability to mono- and poly-ubiquitinate, as well as, ubiquitinateproteins with both lysine-48 and lysine-63 chains has made it difficultto reconcile a common biochemical pathway for parkin's role in PD(Dawson and Dawson, 2010).

A new Parkin Interacting Substrate, PARIS, has been identified, whichprovides a molecular mechanism for neurodegeneration due to parkininactivation in PD. Parkin regulates the levels of PARIS via theubiquitin proteasome system (UPS). PARIS is a major transcriptionalrepressor of peroxisome proliferator-activated receptor gamma (PPARγ)coactivator-1α (PGC-1α) expression and that conditional knockout (KO) ofparkin in adult mice leads to progressive loss of dopamine (DA) neuronsthrough PARIS overexpression and transcriptional repression of PGC-1α.

SUMMARY OF THE INVENTION

A hallmark of Parkinson's disease (PD) is the preferential loss ofsubstantia nigra dopamine neurons. A new Parkin Interacting Substrate,PARIS (ZNF746), is identified whose levels are regulated by theubiquitin proteasome system via binding to and ubiquitination by the E3ubiquitin ligase, parkin. PARIS is a novel KRAB and zinc finger proteinthat accumulates in models of parkin inactivation and in human PD brain.PARIS represses the expression of the transcriptional co-activator,PGC-1α and the PGC-1α target gene, NRF-1 by binding to insulin responsesequences in the PGC-1α promoter. Conditional knockout of parkin inadult animals leads to progressive loss of dopamine (DA) neurons that isPARIS dependent. Moreover overexpression of PARIS leads to the selectiveloss of DA neurons in the substantia nigra, which is reversed by eitherparkin or PGC-1α co-expression. The identification of PARIS provides amolecular mechanism for neurodegeneration due to parkin inactivation.

Applications of the disclosed discoveries include the testing forelevated levels of PARIS or reduced levels of PGC-1α and NRF-1 as adiagnostic test in Parkinson's disease. Any bodily fluid (e.g. urine,blood, cerebra-spinal fluid and brain) or tissue could be tested bymeasuring protein and/or mRNA levels of PARIS, PGC-1α and NRF-1, ortheir metabolites.

PARIS can be used to identify small molecule inhibitors to treatParkinson's disease and related disorders. Additionally, PARIS can beused to identify small molecule inhibitors that leave unaffected otherimportant regulatory signaling of PGC-1α that is PARIS independent.

Reporter constructs for PGC-1α (pGL3-h PGC-1α) are repressed by PARIS.Plus, SK-SHSY cell lines are created to stably express PARIS and pGL3-hPGC-1α and GL3-h PGC-1α alone to be used to screen for PARIS inhibitors.

In vitro and in vivo models of PARIS overexpression and Parkininactivation can be used to validate and optimize the PARIS inhibitors.The disclosed experiments demonstrate the functions of PARIS, whichenables the selection of inhibitors. Positive results will identify themolecules for biologic assays to confirm and characterize PARISinhibitors and to determine their effect on neuronal viability in modelsof Parkinson's disease.

Short hair-pin RNA (shRNA) and anti-sense microRNA inhibitors of PARIScan be used to treat Parkinson's disease and Parkinson's disease relateddisorders.

Inhibitors of PARIS will have broad therapeutic potential onneurodegenerative and related neurologic diseases. Essentially anyindication that has been touted for PPARγ agonists or PGC-1α activatorswill be targets of PARIS inhibition. Some of the neurodegenerative andrelated neurologic diseases are Alexander's disease, Alper's disease,Alzheimer's disease, amyotrophic lateral sclerosis, ataxiatelangiectasia, Batten disease, bovine spongiform encephalopathy,Canavan disease, Cockayne syndrome, corticobasal degeneration,Creutzfeldt-Jakob disease, Huntington's disease, HIV associateddementia, Kennedy's disease, Krabbe's disease, lewy body dementia,Machado-Joseph disease, multiple sclerosis, multiple system atrophy,narcolepsy, neuroborreliosis, Parkinson's disease, Pelizaeus-MerzbacherDisease, Pick's disease, primary lateral sclerosis, prion diseases,Refsum's disease, Sandhoff's disease, Schilder's disease, subacutecombined degeneration of spinal cord secondary to pernicious anemia,schizophrenia, spinocerebellar ataxia, spinal muscular atrophy,Steele-Richardson-Olszewski disease, and tabes dorsalis.

Additionally, inhibitors of PARIS will enable the treatment of a broadspectrum of diseases and disorder. These include:

metabolic disorders, such as, diabetes mellitus, dyslipidemia, andobesity;

atherosclerosis, cardiovascular disease, and cardiac ischemia;

inflammatory conditions, such as, inflammatory bowel diseases, colitisand psoriasis; cancer;

kidney disease, including glomerulonephritis, glomerulosclerosis anddiabetic nephropathy; mitochondrial disorders;

muscle disorders, including muscular dystrophies and

disorders of circadian rhythms and sleep.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent of application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates a summary of the invention.

FIG. 2—Parkin-PARIS-PGC-1α pathway as a model in PD. Endogenous PARISacts to maintain the balance of PGC-1α levels. In PD, parkin isinactivated by diverse insults, such as, familial mutations, reactiveoxygen species (ROS), nitrosative (NO) and dopamine (DA) stress andPARIS accumulates. Accumulated PARIS continuously inhibits PGC-1αtranscription leading to reduction in PGC-1α dependent genes. Ultimatelythis situation results in neurodegeneration in PD. [mean±S.E.M. *p<0.05,**p<0.01, ***p<0.001; ANOVA with the Student-Newman-Keuls post hoctest].

FIG. 3—A regional analysis and levels of PARIS protein expression viaimmunoblot in various brain regions. [mean±S.E.M., n=3].

FIG. 4—Multiple sequence alignment of human (SEQ ID NO. 2), mouse (SEQID NO. 155) and rat (SEQ ID NO. 156) PARIS reveals highly conservedamino acid sequence among the different species. The KRAB domain (bluebar) and zinc finger domains (black bars) are indicated.

FIG. 5—Northern blot analysis of PARIS gene expression in differenttissues. Relative levels of PARIS normalized to GAPDH control isindicated in bottom panel.

FIG. 6—Regional analysis of PARIS protein expression in various mousetissues. Relative levels of PARIS normalized to β-actin control isindicated in bottom panel. Repeated three times with similar results.

FIG. 7—Representative confocal images with (a) anti-parkin antibodyalone, (b) anti-PARIS antibody alone, or (c) anti-parkin along withanti-PARIS antibodies reveal that there is no channel crosstalk and thatendogenous PARIS and parkin are co-localized mostly in the cytoplasm ofSH-SY5Y dopaminergic-like cells. Parkin (green); PARIS (red); Nucleus(DAPI, blue), n=4.

FIG. 8—Immunoblot analysis using a polyclonal PARIS antibody in SH-SY5Ycells transduced with lentiviral shRNA-dsRed or shRNA-PARIS. Thepolyclonal PARIS antibody used in these studies is specific for PARISsince it recognizes a single band on immunoblot from mouse brain.β-actin was used as a loading control, n=3.

FIG. 9—A schematic representation of PARIS. The conserved Kruppelassociated Box (KRAB) and Zinc Finger motifs and their location areindicated.

FIG. 10—Immunohistochemical distribution of PARIS in sagittal andcoronal sections of six-week-old male C57BL mouse brain. Right upperpanel shows an antigen preabsorption control. Ctx, cerebral cortex; Hip,hippocampus; CPu, caudate putamen; SNpr, SN pars reticulata; SNpc, SNpars compacta; Th, thalamus; Cb, cerebellum; (Cb); PC, Purkinje cells;ML, molecular cell layer; GL, granule cell layer; DG, dentate gyrus.Dashed (—) line outlines SN. High power view of SNpc (rectangles inthird row) is shown in the third row middle and right panels and lowermiddle and right panel. Scale bars, 200 μm unless indicated, n=3.

FIG. 11—Confocal microscopy of endogenous PARIS and parkin in thecytoplasm of rat cortical neurons. Top Panel, Left—Parkin; MiddleLeft—PARIS, Middle Right—DAPI, Right—merge of Parkin and Paris images.Inset—high power view of an individual neuron. Bottom panel,Left—Parkin; Middle Left—PARIS, Middle Right—Nucleus-DAPI, Right—merge,n=4.

FIG. 12—Immunoprecipitated (IP) FLAG-PARIS interacts with MYC-Parkin,but not MYC-XIAP or the PARIS homologue, V5-ZNF398 (lane 6), n=3.

FIG. 13—GST-pull down assay between parkin and PARIS indicates a robustinteraction between parkin and PARIS, n=4.

FIG. 14—Immunoprecipitated FLAG-PARIS interacts with WT Parkin andParkin mutants (C431F, G430D, R275W, Q311X) in SH-SY5Y cells. Lowerpanel, relative binding [mean±S.E.M., n=3, *p<0.05, Student's t-test].

FIG. 15—Immunoblot (IB) shows that Parkin and PARIS co-immunoprecipitatein human striatum, n=3.

FIG. 16—Co-immunoprecipitation between parkin and PARIS in mouse brainusing antibodies to parkin, PARIS. Mouse IgG was used as a control.

FIG. 17—Parkin and PARIS co-immunoprecipitate from WT mouse ventralmidbrain, but not parkin KO ventral midbrain, n=3.

FIG. 18—Full-length WT PARIS and the F2 fragment of PARIS interact withparkin.

FIG. 19—PARIS interacts with parkin's RING1 and RING2 domains.Immunoprecipitated MYC-parkin deletions bind FLAG-PARIS except for theIBR domain (lane 8 on left bottom panel).

FIG. 20—Immunoprecipitated FLAG-PARIS interacts with WT and theC-terminal RING domains of parkin, but not the N-terminal UBL-SH domain(bottom right panel).

FIG. 21—WT MYC-Parkin ubiquitinates FLAG-PARIS (lane 3). Parkin mutants(C431F, G430D, and Q311 X) are unable to efficiently ubiquitinateFLAG-PARIS (lanes 4-7). Ubiquitination (Ub(n)) is indicated on rightwith brackets, n=3.

FIG. 22—Endogenous ubiquitination of PARIS (lane 2) is enhanced withexogenous WT Parkin (lane 3) and it is eliminated with shRNA-Parkin(lane 4). In the presence of shRNA-Parkin, robust ubiquitination ofPARIS is observed via co-expression of shRNA-resistant WT parkin(WT^(R)) but not shRNA-resistant Q311X mutant parkin (Q311X^(R)), n=3.

FIG. 23—In vitro ubiquitination reactions with GST-PARIS, E1, E2s UbcH2(2), UbcH3 (3), UbcH5a (5a), UbcH5b (5b), UbcH5c (5c), UbcH6 (6), UbcH7(7), UbcH8 (8), and His-tagged parkin were performed at pH=7.5 showingthat parkin ubiquitinates PARIS in the presence of various E2 enzymeincluding UbcH5c, n=3.

FIG. 24—In vitro ubiquitination reactions with His-tagged parkin, E1,E2s UbcH2 (2), UbcH3 (3), UbcH5a (5a), UbcH5b (5b), UbcH5c (5c), UbcH6(6), UbcH7 (7), UbcH8 (8), in the absence of GST-PARIS were performed atpH=7.5 showing that the ubiquitin signal (FIG. 23) was derived fromPARIS and not parkin, n=3.

FIG. 25—In vitro ubiquitination reactions with E1, E2s UbcH2 (2), UbcH3(3), UbcH5a (5a), UbcH5b (5b), UbcH5c (5c), UbcH6 (6), UbcH7 (7), UbcH8(8), in the absence of GST-PARIS and His-tagged parkin were performed atpH=7.5 showing that there is no ubiquitin signal in the absence ofparkin and PARIS, n=3.

FIG. 26—ChIP, which acts as an E4 for parkin (Imai et al., 2002),enhances the ubiquitination of PARIS by parkin, but it has no affect inthe absence of parkin, n=3.

FIG. 27—OTU1, a K48-linkage specific deubiquitinating enzyme eliminatesthe ubiquitination of PARIS by parkin and ChIP indicating that parkinubiquitinates PARIS via K48 linkages. K48 ubiquitin linkages wereconfirmed by immunoreactivity with a K48-specific anti-ubiquitinantibody (Apu2) and no immunoreactivity with a K63-specificanti-ubiquitin antibody (Apu3), n=3.

FIG. 28—Parkin mediated ubiquitination of FLAG-PARIS in SH-SY5Y cells isvia K48 linkages as co-expression of OTU1 eliminates the ubiquitination,n=3.

FIGS. 29—10 μM MG-132 increases PARIS steady state levels compared toDMSO control. Bottom panel, relative PARIS levels normalized top-actin,n=3. [mean±S.E.M., *p<0.05, **p<0.01 and ***p<0.001, Student's t-test].

FIG. 30—Increasing ratio (1:1 to 4:1) of MYC-Parkin results in decreasedsteady-state levels of FLAG-PARIS (lanes 1-4). Bottom panel, relativePARIS and parkin levels normalized top-actin, n=3; regression analysis,R²=0.9985, p<0.05).

FIG. 31—WT Parkin decreases the steady-state levels of PARIS compared tomutant Q311X parkin or GFP transfected control cells in cyclohexamide(CHX)-chase experiments in SH-SY5Y cells transiently expressingFLAG-PARIS. Bottom panel, relative PARIS levels normalized to β-actin,n=3. [mean±S.E.M., *p<0.05, **p<0.01 and ***p<0.001, ANOVA withStudent-Newman-Keuls post-hoc analysis].

FIG. 32—MYC-parkin leads to degradation of FLAG-PARIS at a 4 to 1 ratio,respectively. 10 μM MG-132 prevents the degradation of FLAG-PARIS andMYC-Q311X parkin has no effect, n=3). [mean±S.E.M., *p<0.05, **p<0.01and ***p<0.001, ANOVA with Student-Newman-Keuls post-hoc analysis].

FIG. 33—PARIS accumulates after shRNA-Parkin and co-expression of shRNAresistant parkin (MYC-Parkin^(R)) leads to robust degradation of PARIS,n=3. [Data=mean±S.E.M., *p<0.05, **p<0.01 and ***p<0.001, ANOVA withStudent-Newman-Keuls post-hoc analysis].

FIG. 34—Immunoblot analysis of PARIS and β-actin in cingulate cortexfrom age-matched controls and AR-PD patient brains with parkinmutations.

FIG. 35—Quantitation of the immunoblots in FIG. 34 normalized toβ-actin, n=4. [Data=mean±S.E.M., *p<0.05, **p<0.01 and ***p<0.001,unpaired two-tailed Student's t-test].

FIG. 36—PARIS levels in cerebellum (CBM), frontal cortex (FC), striatum(STR) and SN of sporadic PD patient brains compared to age-matchedcontrols.

FIG. 37—Relative PARIS levels normalized to 8-actin in FIG. 35, Controlsn=4; PD n=5. [Data=mean±S.E.M., *p<0.05, **p<0.01 and ***p<0.001, ANOVAtest with Student-Newman-Keuls post-hoc analysis].

FIG. 38—Real-time qRT-PCR of IRS (PEPCK-like motif)-containing genes andPGC-1α dependent genes in PD SN compared to age-matched CTL-SNnormalized to GAPDH, n=3-4 per group. [mean±S.E.M., *p<0.05, **p<0.01and ***p<0.001, unpaired two-tailed Student's t-test].

FIG. 39—Real-time qRT-PCR of qRT-PCR of IRS (PEPCK-likemotif)-containing genes and PGC-1α dependent genes reveals that PGC-1αand NRF-1 are reduced in PD striatum compared to age-matched controls(n=4 per group). Relative mRNA levels of PGC-1α, parkin, PARIS andPGC-1α dependent genes normalized to GAPDH are indicated. No mRNA forAPOC3 and TAT was detected with 2 different pairs of primers.

FIG. 40—ChIP assay of endogenous PARIS binding to the IRS region of thehuman PGC-1α promoter in human PD and aged-matched control (CTL)striatum, control n=3; PD n=4.

FIG. 41—Quantitation of ChIP in FIG. 40. Human specific IRS primers forFIG. 67 and FIG. 40 are indicated in FIG. 57. [mean±S.E.M., *p<0.05,**p<0.01, ***p<0.001, unpaired two-tailed Student's t-test].

FIG. 42—Relative mRNA levels as determined by real-time qRT-PCR of IRS(PEPCK-like motif)-containing genes, PGC-1α, PARIS, parkin and PGC-1αdependent genes normalized to GAPDH in the SN of the lentiviral-mediatedconditional parkin knockout model, n=4 per group. See Table 4 forqRT-PCR primers.

FIG. 43—Immunoblots of PARIS, PGC-1α, parkin and NRF-1 in soluble andinsoluble fractions of PD SN compared to age-matched CTL-SN.

FIG. 44—Quantitation of the immunoblots in FIG. 43 normalized toβ-actin, PD, n=4; Control, n=3. [mean±S.E.M., *p<0.05, **p<0.01 and***p<0.001, unpaired two-tailed Student's t-test].

FIG. 45—Immunoblot analysis shows that PGC-1α and NRF-1 protein levelsare reduced in PD striatum compared to age-matched controls, n=4 pergroup. Parkin redistributes to the insoluble fraction consistent withits inactivation in PD.

FIG. 46—Quantitation of the immunoblots in FIG. 45 normalized toβ-actin, n=4 per group. (See Table 5 for details on human brainsamples).

FIG. 47—a-f, Regression analysis of the quantified level of PARIS,PGC-1α, and NRF-1 shows there is a strong negative correlation betweenthe protein levels of PARIS and PGC-1α (R²=0.5195, p<0.05) and NRF-1(R²=0.8015, p<0.01) in the striatum and between PARIS and PGC-1α(R²=0.6955, p<0.05) and NRF-1 (R²=0.5979, p<0.05) in the SN and apositive correlation between PGC-1α and NRF-1 in the striatum(R²=0.6827, p<0.05) and in the SN(R²=0.6488, p<0.05), n=4 per group. SeeTable 5 for details on human brain samples.

FIG. 48—PARIS occupies the endogenous PEPCK and G6Pase promoter. ChIPassay shows increased endogenous binding of PARIS in human PD andaged-matched control striatum to the IRS region of the human PGC-1αpromoter and occupies the PEPCK and G6Pase promoter (CTL n=3; PD n=4;see Table 5 for details on human brain samples).

FIG. 49—Immunoblot analysis of PARIS in cortex (CTX), STR and ventralmidbrain (VM) from WT and parkin exon 7 KO 18-24 month old mice.

FIG. 50—Relative protein levels of PARIS normalized to β-actin for FIG.49, WT n=9; parkin KO n=10. [Data=mean±S.E.M., *p<0.05, **p<0.01 and***p<0.001, ANOVA test with Student-Newman-Keuls post-hoc analysis].

FIG. 51—PARIS mRNA levels in indicated brain regions from WT and parkinexon 7 KO 18-24 month old mice. [Data=mean±S.E.M., *p<0.05, **p<0.01 and***p<0.001, ANOVA test with Student-Newman-Keuls post-hoc analysis].

FIG. 52—Top panel, experimental illustration of stereotaxic intranigralvirus injection. Bottom panels, immunofluorescent images of TH, GFP andmerged in exon 7 floxed parkin mice (parkin^(Flx/Flx)) afterstereotactic delivery of Lenti-GFP or Lenti-GFPCre into the SNpc.84.9±1.9% and 78.1±2.6% of TH neurons express GFP and GFPCre,respectively, n=3 per group. Enlarged images in the right bottom panelswere taken from the white rectangle region from the merged images ofLenti-GFPCre and Lenti-GFP, bar=100 μm.

FIG. 53—Immunoblot analysis of parkin, PARIS, actin and GFP 4 weeksafter intranigral Lenti-GFPCre or Lenti-GFP injection intoparkin^(Flx/Flx) mice.

FIG. 54—Relative protein levels of PARIS normalized to β-actin for FIG.53. [Data=mean±S.E.M., *p<0.05, **p<0.01 and ***p<0.001, unpairedtwo-tailed Student's t-test].

FIG. 55—GAL4-luciferase assay demonstrates that PARIS decreases promoteractivity, which is recovered by co-expression of WT parkin, but notQ311X mutant. Relative luciferase activity compared to Renillaluciferase control is indicated in the histogram (n=3). Immunoblotanalysis confirms the expression of FLAG-PARIS, MYC-Parkin, andMYC-Q311X parkin. β-Actin was used as a loading control (bottom ofpanel). *p<0.05, **p<0.01 in an ANOVA test followed byStudent-Newman-Keuls post-hoc analysis. Schematic representation of thepromoter construct is indicated at the top of the panel.

FIG. 56—Identification and MACAW alignment of the PARIS DNA-bindingsequence as determined by CASTing. Darker shades represent a greaterdegree of overlap of the segment pairs (bottom right—% overlap).*Duplicate sequence tags.

FIG. 57—Relative luciferase activity of the 1-kilobase human PGC-1α(−992 to +90) compared to Renilla luciferase±PARIS or ±parkin or±familial mutant Q311X parkin, n=3. Location of IRS, CRE motifs andoligos for human ChIP (arrows) in the PGC-1α promoter construct (top ofpanel). Immunoblot analysis confirms the expression of FLAG-PARIS,MYC-Parkin and MYC-Q311X parkin (right panel). [mean±S.E.M., *p<0.05,**p<0.01, ***p<0.001, ANOVA with Student-Newman-Keuls post-hocanalysis].

FIG. 58—PARIS represses the mouse PGC-1α promoter luciferase reporteractivity. Co-transfection of SH-SY5Y cells with PARIS and the mousePGC-1α promoter (−2533 to +78), results in decreased activity of PGC-1αpromoter. Introduction of WT parkin rescues PARIS suppression of themouse PGC-1α promoter. Relative luciferase activity compared to Renillaluciferase control is indicated in histogram (n=4). Immunoblot analysisconfirms the expression of FLAG-PARIS, MYC-Parkin and β-actin was usedas a loading control (bottom of panel). *p<0.05 in an ANOVA testfollowed by Student-Newman-Keuls post-hoc analysis. Schematicrepresentation of the promoter construct is indicated at the top of thepanel.

FIG. 59—EMSA of GST-PARIS of ³²P-labeled WT (WT-³²P) IRSoligonucleotides (IRS1-WT (SEQ ID NO. 8), IRS2-WT (SEQ ID NO. 10) andIRS3-WT (SEQ ID NO. 12)) of the human PGC-1α promoter and ³²P-labeledmutant (T-g) (MT-³²P) IRS oligonucleotides (IRS1-MT (SEQ ID NO. 9),IRS2-MT (SEQ ID NO. 11) and IRS3-MT (SEQ ID NO. 13)). Unlabeled WT (WTcold) IRS oligonucleotides disrupt the GST-PARIS-DNA protein complexeswith the WT-³²P IRS oligonucleotides, n=3. Unlabeled mutant probe (MTcold) has no effect on mutant (MT-³²P). Arrow indicates specificPARIS-shifted probe; NS, nonspecific; FS, fragmented PARIS-shiftedprobe; FP, free probe.

FIG. 60—Schematic representation of the PGC-1α WT, IRS1-M, IRS2-M,IRS3-M and IRS123-M promoter reporter constructs harboring the T→G pointmutation on the IRS elements is indicated at the top of the panel. Thebasal promoter activities of PGC-1α WT, IRS1-M, IRS2-M, IRS3-M andIRS123-M promoter reporter constructs were monitored in the presence andabsence of PARIS. Relative luciferase activity compared to Renillaluciferase control is indicated in histogram (n=3). *p<0.05 in an ANOVAtest followed by Student-Newman-Keuls post-hoc analysis. Immunoblotanalysis confirms the expression of FLAG-PARIS. β-actin was used as aloading control.

FIG. 61—Schematic representation of PARIS zinc finger mutants.

FIG. 62—The basal promoter activity of the human PGC-1α promoterreporter construct in SH-SY5Y cells was monitored in the presence andabsence of WT PARIS and the 8 zinc finger PARIS mutants depicted in FIG.55. Relative luciferase activity compared to Renilla luciferase controlis indicated in the histogram, n=3. Immunoblot analysis confirms theexpression of GFP-PARIS, and PARIS zinc finger mutants. β-actin was usedas a loading control (Top Panel). *p<0.05 in an ANOVA test followed byStudent-Newman-Keuls post-hoc analysis.

FIG. 63—EMSA reveals that GST-PARIS binds to ³²P-labeled WT (WT-³²P) IRSoligonucleotides (IRS1-WT, IRS2-WT, IRS3-WT) of the human PGC-1αpromoter, whereas the PARIS C571A mutant has substantially reducedbinding (n=3). The arrow indicates the specific PARIS-shifted probe. NS,nonspecific; FS, fragmented PARIS-shifted probe; FP, free probe.

FIG. 64—Real-time qRT-PCR of PGC-1α, GFP and β-actin following transienttransfection of GFP, GFP-PARIS or GFP-0571A PARIS mutant, n=4.[mean±S.E.M., *p<0.05, **p<0.01, ***p<0.001, ANOVA withStudent-Newman-Keuls post-hoc analysis].

FIG. 65—Immunoblot analysis of PGC-1α, GFP and β-actin followingtransient transfection of GFP, GFP-PARIS or GFP-0571A PARIS mutant, n=4.

FIG. 66—Quantitation of the immunoblots in FIG. 65 normalized toβ-actin, n=4. [mean±S.E.M., *p<0.05, **p<0.01, ***p<0.001, ANOVA withStudent-Newman-Keuls post-hoc analysis].

FIG. 67—PARIS occupies the endogenous PGC-1α promoter as determined byChIP assay with anti-PARIS polyclonal antibodies in SH-SY5Y cells, n=3.

FIG. 68—PARIS occupies the endogenous mouse PGC-1α promoter asdetermined by ChIP in mouse whole brain, n=3. Mouse specific IRS primersare indicated in FIG. 58.

FIG. 69—Schematic representation of the promoter construct of humanPEPCK.

FIG. 70—Schematic representation of the promoter construct of humanG6Pase, including a representation of IRS1′, IRS2′, and IRS3′. FIG. 70discloses the full-length sequence as SEQ ID NO: 154.

FIG. 71—PARIS does not repress the IRS-containing genes, PEPCK andG6Pase. Co-transfection of SH-SY5Y cells with PARIS and the rat PEPCK(rPEPCK) promoter-luciferase reporter (−2100 to +68), results inincreased promoter activity of rPEPCK. While overexpression of PARISdoes not alter mouse G6Pase (mG6Pase) promoter (−484 to +66) activity.Relative luciferase activity compared to Renilla luciferase control isindicated in histogram, n=3. Immunoblot analysis confirms the expressionof FLAG-PARIS and β-actin was used as a loading control (top of panel).***p<0.001 in an unpaired two-tailed Student's t-test.

FIG. 72—PARIS occupies the endogenous PEPCK and G6Pase promoter. ChIPassay monitoring the occupancy of the endogenous PEPCK and G6Pasepromoter by PARIS in SH-SY5Y, n=3.

FIG. 73—Immunoblot analysis of parkin, PARIS, PGC-1α and β-actin indouble knockdown experiments via lentiviral transduction of shRNA-parkinand/or shRNA-PARIS in SH-SY5Y cells, n=3.

FIG. 74—Quantitation of the immunoblots in FIG. 73 normalized toβ-actin, n=3, sh=shRNA. [mean±S.E.M., *p<0.05, **p<0.01, ***p<0.001,ANOVA with Student-Newman-Keuls post-hoc analysis].

FIG. 75—Relative mRNA levels of PGC-1α normalized to GAPDH, n=3.[mean±S.E.M., *p<0.05, **p<0.01, ***p<0.001, ANOVA withStudent-Newman-Keuls post-hoc analysis].

FIG. 76—Top panel, experimental illustration of stereotaxic intranigralvirus injection. Below are immunoblots of parkin, PARIS, PGC-1α, NRF-1,β-actin and GFP, 4 weeks after stereotactic delivery of Lenti-GFP,Lenti-GFPCre, Lenti-GFPCre+shRNA-dsRed, or Lenti-GFPCre+shRNA-PARIS intoexon 7 floxed parkin mice (parkin^(Flx/Flx)), n=3 per group.*nonspecific band.

FIG. 77—Quantitation of the immunoblots in FIG. 76 normalized toβ-actin, n=3 per group. [mean±S.E.M., *p<0.05, **p<0.01 and ***p<0.001,ANOVA with Student-Newman-Keuls post-hoc analysis].

FIG. 78—The alteration of PGC-1α and NRF1 shown in FIG. 76 and FIG. 77results from transcriptional changes as determined by real-time qRT-PCR,n=3 per group. [mean±S.E.M., *p<0.05, **p<0.01 and ***p<0.001, ANOVAwith Student-Newman-Keuls post-hoc analysis].

FIG. 79—Stereological assessment of TH and NissI positive neurons in theSN of parkin^(Flx/Flx) mice injected with Lenti-GFP, andLenti-GFPCre±Lenti-shRNA-PARIS 3 (n=3 per group) and 10 months (n=7 pergroup) after injection of virus. [mean±S.E.M., *p<0.05, **p<0.01 and***p<0.001, ANOVA with Student-Newman-Keuls post-hoc analysis].

FIG. 80—Representative photomicrographs of laser capture microdissection(LCM) of dopaminergic neurons from conditional parkin KO mice. Upperpanels are immunofluorescent images of TH and either GFP (left panel) orGFPCre (right panel) in conditional parkin KO mice before LCM. Lowerpanels are after LCM.

FIG. 81—Robust reduction of PGC-1α mRNA in microdissected parkin KOdopaminergic neurons. PARIS level was assessed to monitor RNA quality(negative control). Values were normalized to GAPDH, n=3 per group.***p<0.001 in an unpaired two-tailed Student's t-test.

FIG. 82—PGC-1α and NRF-1 levels are not altered in the ventral midbrain(VM) of 18-24 month old germline parkin exon 7 KO mice compared toage-matched WT controls mice where is PARIS is modestly upregulated, WT,n=4; KO, n=5.

FIG. 83—Quantitation of the immunoblots in FIG. 82 normalized toβ-actin, WT, n=4; KO, n=5. All data are expressed as mean±S.E.M.Asterisk indicates statistical significance (*p<0.05, **p<0.01 and***p<0.001) in an unpaired two-tailed Student's t-test.

FIG. 84—Top panel, experimental illustration of stereotaxic intranigralvirus injection. Below is TH immunostaining of representative micemidbrain sections from SN of parkin^(Flx/Flx) mice injected withLenti-GFP, and Lenti-GFPCre±Lenti-shRNA-PARIS 10 months post-injectionof virus.

FIG. 85—Schematic illustration of intranigral viral injection andtransduced brain regions.

FIG. 86—Immunoblot analysis of PARIS, PGC-1α, parkin and NRF-1four-weeks post intranigral injection of AAV1-PARIS, n=5 per group.

FIG. 87—Quantitation of the immunoblots in FIG. 22 normalized toβ-actin.

FIG. 88—TH staining of a representative section of mice injected withAAV1-GFP, AAV1-PARIS±AAV1-parkin or AAV1-PARIS±Lenti-PGC-1α. Each panelshows the noninjected side (Non) and contralateral injected side (Inj)and white pentagonal box indicates the SNpc. Enlarged images containingSNpc and SNpr are shown on the right panels. AAV1 encoding GFP was usedas transduction control in all injection procedures. Broad regionsincluding SNpc and SNpr were successfully transduced (left top panel).In right bottom panel, yellow rectangle indicates the region that PARISand lenti-PGC-1α co-transduced. Approximately 30% of the SNpc wastransduced with lenti-PGC-1α and this is the region, which is protectedfrom PARIS toxicity, n=6 per group.

FIG. 89—Stereological TH, NissI-positive neuronal counting, n=6 pergroup.

FIG. 90—Representative SN sections stained with a-GAD 65/67, Robustviral expression was evaluated by GFP immunofluoresence in SNpr (toppanel). The GABAergic neuronal marker shows no difference betweenAAV1-GFP and AAV1-PARIS between noninjected side and injected side, n=6per group.

FIG. 91—Equivalent protein levels of GAD 65/67 were confirmed byimmunoblot analysis, n=5 per group.

FIG. 92—Quantitation of the immunoblots in FIG. 91 normalized toβ-actin, n=5. Data are expressed as mean±S.E.M. Statistical significancewas evaluated ANOVA with the Student-Newman-Keuls post hoc test.

FIG. 93—Immunoblot analysis of PARIS, PGC-1α, parkin and NRF-1, n=3.

FIG. 94—Quantitation of the immunoblots in FIG. 32 normalized toβ-actin.

FIGS. 2, 85-89, 93 and 94 illustrate introduction of AAV1-Parkin orLenti-PGC-1α in mice SN protects from AAV1-PARIS-mediated selectivedopaminergic neuronal toxicity.

FIGS. 3 and 9-11 illustrate the identification of a novel parkininteracting substrate, PARIS.

FIGS. 4-8 illustrate characterization of PARIS.

FIGS. 12-15 and 17 illustrate Parkin interacts with PARIS.

FIGS. 16 and 18-20 illustrates Protein interaction mapping betweenparkin and PARIS.

FIGS. 21, 22 and 29-33 illustrate Parkin ubiquitinates and regulates theubiquitin proteasomal degradation of PARIS.

FIGS. 23-28 illustrate Parkin ubiquitinates PARIS in vitro.

FIGS. 30, 41, 56, 57, 59, 64, 66-68, 74 and 75 illustrate PARIS acts astranscriptional repressor of PGC-1α.

FIGS. 34-37 illustrate PARIS accumulates in AR-PD, sporadic PD and inanimal models of parkin inactivation.

FIGS. 38, 43, 44, 76-79 and 84 illustrate identification of PGC-1α andNRF-1 as pathological in vivo targets of accumulated PARIS in PD brainand conditional parkin KO mice.

FIGS. 39, 42, 45-47 and 80-83 illustrate identification of PGC-1α andNRF-1 as in vivo targets of accumulated PARIS in PD brain.

FIGS. 48, 55, 58, 60-63 and 69-72 illustrate PARIS is a transcriptionalrepressor.

FIGS. 90-92 illustrates lack of degeneration of GABAergic neurons.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for an elevated level of PARIS.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for an elevated level of PARIS in urine.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for an elevated level of PARIS in blood.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for an elevated level of PARIS incerebra-spinal fluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for an elevated level of PARIS in thebrain.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for an elevated level of PARIS in abodily fluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of PARIS.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of PARIS in urine.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of PARIS in blood.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of PARIS incerebra-spinal fluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of PARIS in the brain.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of PARIS in a bodilyfluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for an elevated level of a metabolite ofPARIS.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for an elevated level of a metabolite ofPARIS in urine.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for an elevated level of a metabolite ofPARIS in blood.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for an elevated level of a metabolite ofPARIS in cerebra-spinal fluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for an elevated level of a metabolite ofPARIS in the brain.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for an elevated level of a metabolite ofPARIS in a bodily fluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of a metabolite ofPARIS.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of a metabolite ofPARIS in urine.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of a metabolite ofPARIS in blood.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of a metabolite ofPARIS in cerebra-spinal fluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of a metabolite ofPARIS in the brain.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of a metabolite ofPARIS in a bodily fluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring level of an mRNA coding for PARIS.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring level of an mRNA coding for PARIS inurine.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring level of an mRNA coding for PARIS inblood.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring level of an mRNA coding for PARIS incerebra-spinal fluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring level of an mRNA coding for PARIS inblood the brain.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring level of a mRNA coding for PARIS in abodily fluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for a reduced level of PGC-1α.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for a reduced level of PGC-1α in urine.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for a reduced level of PGC-1α in blood.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for a reduced level of PGC-1α incerebra-spinal fluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for a reduced level of PGC-1α in thebrain.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by testing for a reduced level of PGC-1α in a bodilyfluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of PGC-1α.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of PGC-1α in urine.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of PGC-1α in blood.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of PGC-1α incerebra-spinal fluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of PGC-1α in the brain.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring a protein level of PGC-1α in a bodilyfluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring level of an mRNA coding for PGC-1α.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring level of an mRNA coding for PGC-1α inurine.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring level of an mRNA coding for PGC-1α inblood.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring level of an mRNA coding for PGC-1α incerebra-spinal fluid.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring level of an mRNA coding for PGC-1α inthe brain.

In one embodiment the invention is drawn to a method to diagnoseParkinson's disease by measuring level of an mRNA coding for PGC-1α in abodily fluid.

In one embodiment the invention is drawn to a method to identify smallmolecular compound that can be used to treat Parkinson's disease.

In one embodiment the invention is drawn to a method to identify smallmolecular compound that can be used to treat Parkinson's disease relateddisorders.

In one embodiment the invention is drawn to a reporter construct forPGC-1α (pGL3-h PGC-1α) that is repressed by PARIS.

In one embodiment the invention is drawn to a SK-SHSY cell line tostably express PARIS and pGL3-h PGC-1α, and GL3-h PGC-1α alone, whereinPARIS and pGL3-h PGC-1α are used to screen for PARIS inhibitors.

In one embodiment the invention is drawn to a method to identify a smallmolecule inhibitor of PARIS that leave unaffected other regulatorysignaling of PGC-1α that is PARIS independent.

In one embodiment the invention is drawn to an in vitro model of PARISoverexpression that can be used to validate a PARIS inhibitor.

In one embodiment the invention is drawn to an in vitro model of PARISoverexpression that can be used to optimize a PARIS inhibitor.

In one embodiment the invention is drawn to an in vivo model of PARISoverexpression that can be used to validate a PARIS inhibitor.

In one embodiment the invention is drawn to an in vivo model of PARISoverexpression that can be used to optimize a PARIS inhibitor.

In one embodiment the invention is drawn to an in vitro model of Parkininactivation that can be used to validate a PARIS inhibitor.

In one embodiment the invention is drawn to an in vitro model of Parkininactivation that can be used to optimize a PARIS inhibitor.

In one embodiment the invention is drawn to an in vivo model of Parkininactivation that can be used to validate a PARIS inhibitor.

In one embodiment the invention is drawn to an in vivo model of Parkininactivation that can be used to optimize a PARIS inhibitor.

In one embodiment the invention is drawn to a method to select aninhibitor of PARIS by disrupting a function of PARIS.

In one embodiment the invention is drawn to a method to develop abiologic assay to confirm and/or characterize a PARIS inhibitor.

In one embodiment the invention is drawn to a method to determine theeffect of a PARIS inhibitor on neuronal viability in models ofParkinson's disease.

In one embodiment the invention is drawn to an isolated nucleotide ofSEQ ID NO. 27.

In one embodiment the invention is drawn to an isolated nucleotide ofSEQ ID NO. 28.

In one embodiment the invention is drawn to a method of treatingParkinson's disease by administering a shRNA inhibitor.

In one embodiment the invention is drawn to a method of treatingParkinson's disease by administering an anti-sense microRNA inhibitor.

In one embodiment the invention is drawn to a method of treatingParkinson's disease related disorders by administering a shRNAinhibitor.

In one embodiment the invention is drawn to a method of treatingParkinson's disease related disorders by administering an anti-sensemicroRNA inhibitor.

In one embodiment the invention is drawn to a method of treatingneurodegenerative and related neurologic diseases, such as, Alexander'sdisease, Alper's disease, Alzheimer's disease, amyotrophic lateralsclerosis, ataxia telangiectasia, Batten disease, bovine spongiformencephalopathy, Canavan disease, Cockayne syndrome, corticobasaldegeneration, Creutzfeldt-Jakob disease, Huntington's disease, HIVassociated dementia, Kennedy's disease, Krabbe's disease, lewy bodydementia, Machado-Joseph disease, multiple sclerosis, multiple systematrophy, narcolepsy, neuroborreliosis, Parkinson's disease,Pelizaeus-Merzbacher Disease, Pick's disease, primary lateral sclerosis,prion diseases, Refsum's disease, Sandhoff's disease, Schilder'sdisease, subacute combined degeneration of spinal cord secondary topernicious anemia, schizophrenia, spinocerebellar ataxia, spinalmuscular atrophy, Steele-Richardson-Olszewski disease, and tabesdorsalis; by administering an inhibitor of PARIS.

In one embodiment the invention is drawn to a method of treatingmetabolic disorders such as diabetes mellitus, dyslipidemia, andobesity, by administering am inhibitor of PARIS.

In one embodiment the invention is drawn to a method of treating acirculatory disorder, such as, atherosclerosis, cardiovascular disease,and cardiac ischemia, by administering an inhibitor of PARIS.

In one embodiment the invention is drawn to a method of treatinginflammatory conditions such as inflammatory bowel diseases, colitis andpsoriasis; by administering an inhibitor of PARIS.

In one embodiment the invention is drawn to a method of treatingtreatment of cancer by administering an inhibitor of PARIS.

In one embodiment the invention is drawn to a method of treating kidneydiseases, including glomerulonephritis, glomerulosclerosis and diabeticnephropathy; by administering an inhibitor of PARIS.

In one embodiment the invention is drawn to a method of treatingmitochondrial disorders by administering an inhibitor of PARIS.

In one embodiment the invention is drawn to a method of treating muscledisorders, including muscular dystrophies, by administering an inhibitorof PARIS.

In one embodiment the invention is drawn to a method of treatingdisorders of circadian rhythms and sleep by administering an inhibitorof PARIS.

In one embodiment the invention is drawn to an isolated polypeptidecomprising of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to an isolated polypeptideconsisting of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to an isolated polypeptidecomprising a peptide sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to an isolated polypeptidecomprising the peptide sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to an isolated polypeptidecomprising a polypeptide coded by the nucleotide sequence of any of SEQID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to an isolated polynucleotidecomprising of any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to an isolated polynucleotideconsisting of any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to an isolated polynucleotidecomprising a nucleotide sequence of any of SEQ ID NO: 5 to SEQ ID NO:136.

In one embodiment the invention is drawn to an isolated polynucleotidecomprising the nucleotide sequence of any of SEQ ID NO: 5 to SEQ ID NO:136.

In one embodiment the invention is drawn to an isolated polynucleotidecomprising a polynucleotide encoding the amino acid sequence of any ofSEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to an isolated polynucleotidecomprising a polynucleotide which hybridizes under stringent conditionsto any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a composition comprising anisolated polypeptide comprising of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a composition comprising anisolated polypeptide consisting of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a composition comprising anisolated polypeptide comprising a peptide sequence of any of SEQ ID NO:1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a composition comprising anisolated polypeptide comprising the peptide sequence of any of SEQ IDNO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a composition comprising anisolated polypeptide comprising a polypeptide coded by the nucleotidesequence of any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a composition comprising anisolated polynucleotide comprising of any of SEQ ID NO: 5 to SEQ ID NO:136.

In one embodiment the invention is drawn to a composition comprising anisolated polynucleotide consisting of any of SEQ ID NO: 5 to SEQ ID NO:136.

In one embodiment the invention is drawn to a composition comprising anisolated polynucleotide comprising a nucleotide sequence of any of SEQID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a composition comprising anisolated polynucleotide comprising the nucleotide sequence of any of SEQID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a composition comprising anisolated polynucleotide comprising a polynucleotide encoding the aminoacid sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a composition comprising anisolated polynucleotide comprising a polynucleotide which hybridizesunder stringent conditions to any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a composition consisting ofan isolated polypeptide comprising of any of SEQ ID NO: 1 to SEQ ID NO:4.

In one embodiment the invention is drawn to a composition consisting ofan isolated polypeptide consisting of any of SEQ ID NO: 1 to SEQ ID NO:4.

In one embodiment the invention is drawn to a composition consisting ofan isolated polypeptide comprising a peptide sequence of any of SEQ IDNO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a composition consisting ofan isolated polypeptide comprising the peptide sequence of any of SEQ IDNO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a composition consisting ofan isolated polypeptide comprising a polypeptide coded by the nucleotidesequence of any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a composition consisting ofan isolated polynucleotide comprising of any of SEQ ID NO: 5 to SEQ IDNO: 136.

In one embodiment the invention is drawn to a composition consisting ofan isolated polynucleotide consisting of any of SEQ ID NO: 5 to SEQ IDNO: 136.

In one embodiment the invention is drawn to a composition consisting ofan isolated polynucleotide comprising a nucleotide sequence of any ofSEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a composition consisting ofan isolated polynucleotide comprising the nucleotide sequence of any ofSEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a composition consisting ofan isolated polynucleotide comprising a polynucleotide encoding theamino acid sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a composition consisting ofan isolated polynucleotide comprising a polynucleotide which hybridizesunder stringent conditions to any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of an isolated polypeptide comprising of any of SEQ IDNO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of an isolated polypeptide consisting of any of SEQ IDNO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of an isolated polypeptide comprising a peptidesequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of an isolated polypeptide comprising the peptidesequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of an isolated polypeptide comprising a polypeptidecoded by the nucleotide sequence of any of SEQ ID NO: 5 to SEQ ID NO:136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of an isolated polynucleotide comprising of any of SEQID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of an isolated polynucleotide consisting of any of SEQID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of an isolated polynucleotide comprising a nucleotidesequence of any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of an isolated polynucleotide comprising the nucleotidesequence of any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of an isolated polynucleotide comprising apolynucleotide encoding the amino acid sequence of any of SEQ ID NO: 1to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of an isolated polynucleotide comprising apolynucleotide which hybridizes under stringent conditions to any of SEQID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition comprising an isolated polypeptidecomprising of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition comprising an isolated polypeptideconsisting of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition comprising an isolated polypeptidecomprising a peptide sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition comprising an isolated polypeptidecomprising the peptide sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition comprising an isolated polypeptidecomprising a polypeptide coded by the nucleotide sequence of any of SEQID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition comprising an isolated polynucleotidecomprising of any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition comprising an isolated polynucleotideconsisting of any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition comprising an isolated polynucleotidecomprising a nucleotide sequence of any of SEQ ID NO: 5 to SEQ ID NO:136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition comprising an isolated polynucleotidecomprising the nucleotide sequence of any of SEQ ID NO: 5 to SEQ ID NO:136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition comprising an isolated polynucleotidecomprising a polynucleotide encoding the amino acid sequence of any ofSEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition comprising an isolated polynucleotidecomprising a polynucleotide which hybridizes under stringent conditionsto any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition consisting of an isolated polypeptidecomprising of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition consisting of an isolated polypeptideconsisting of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition consisting of an isolated polypeptidecomprising a peptide sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a composition consisting ofan isolated polypeptide comprising the peptide sequence of any of SEQ IDNO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition consisting of an isolated polypeptidecomprising a polypeptide coded by the nucleotide sequence of any of SEQID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition consisting of an isolatedpolynucleotide comprising of any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition consisting of an isolatedpolynucleotide consisting of any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition consisting of an isolatedpolynucleotide comprising a nucleotide sequence of any of SEQ ID NO: 5to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition consisting of an isolatedpolynucleotide comprising the nucleotide sequence of any of SEQ ID NO: 5to SEQ ID NO: 136.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition consisting of an isolatedpolynucleotide comprising a polynucleotide encoding the amino acidsequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment the invention is drawn to a method of treatment forParkinson's disease, comprising administering a pharmaceuticallyeffective amount of a composition consisting of an isolatedpolynucleotide comprising a polynucleotide which hybridizes understringent conditions to any of SEQ ID NO: 5 to SEQ ID NO: 136.

In one embodiment the invention is drawn to a kit comprising at leastone isolated polynucleotide selected from the group consisting ofsequences SEQ ID NO: 5 to SEQ ID NO: 136 and instructions on their use.

In one embodiment the invention is drawn to a kit comprising at leastone isolated polypeptide selected from the group consisting of sequencesSEQ ID NO: 1 to SEQ ID NO: 4 and instructions on their use.

In one embodiment the invention is drawn to a kit comprising acomposition of at least one isolated polypeptide selected from the groupconsisting of sequences SEQ ID NO: 1 to SEQ ID NO: 4 and instructions ontheir use.

In one embodiment the invention is drawn to a kit comprising acomposition of at least one isolated polynucleotide selected from thegroup consisting of sequences SEQ ID NO: 5 to SEQ ID NO: 136 andinstructions on their use.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Forexample, although the above description relates to human cells, variousaspects of the invention might also be applied to cells from othermammals by making appropriate modifications to the described methods.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

DETAILED DESCRIPTION OF THE INVENTION PARIS (ZNF746) is a Novel ParkinSubstrate

PARIS fulfilled several criteria for a parkin substrate and in theabsence of parkin activity in PD it accumulated making it an attractivepathogenic substrate (FIG. 1). In sporadic PD, PARIS only accumulated inthe striatum and SN. The selective inactivation of parkin in thestriatum and SN in sporadic PD through nitrosative and dopaminergicstress and c-Abl phosphorylation most likely accounted for theaccumulation of PARIS. Similarly, in parkin exon 7 KO mice, PARIS wasonly upregulated in the striatum and SN. However, PARIS accumulated inthe cingulate cortex of AR-PD brains, suggesting that parkin regulatedthe expression of PARIS outside the striatum and SN. The lack of anupregulation in the cortex of parkin exon 7 mice, suggested differentialand selective regulatory mechanisms in the cortex versus the striatumand SN.

PARIS is a Novel Transcriptional Repressor of PGC-1α

PARIS is a member of the family of KRAB zinc-finger proteins (KRAB-ZFPs)transcriptional repressors. Homologues of PARIS exist in simplerorganisms suggesting that PARIS function may be evolutionarilyconserved. PARIS seemed to bind exclusively to IRS/PLM motifs, whichprovided an important site of regulation of a variety of IRS/PLMresponsive proteins. PGC-1α levels were revealed as potently andselectively regulated by PARIS, consistent with the observation thatPGC-1α transcription was controlled by IRS/PLM motifs. In PD SN andstriatum and conditional parkin KO midbrain, PGC-1α was the only IRS/PLMregulated gene that was downregulated suggesting that PARIS selectivelyrepressed PGC-1α expression in PD.

Notably PARIS was a physiological transcriptional repressor of PGC-1α,which directly and endogenously occupied the cis-regulatory elements ofPGC-1α. PGC-1α was a transcriptional co-activator that controlled thetranscription of many genes involved in cellular metabolism includingmitochondrial biogenesis and respiration and ROS metabolism. The levelsof PGC-1α dependent genes were controlled in large part by the natureand composition of the PGC-1α transcriptional co-activator complex. ThePGC-1α dependent gene, NRF-1, appeared to be particularly susceptible tothe inhibitory effects of PARIS.

It was likely that there was not only a PARIS transcriptional repressorprotein complex that played important regulatory roles in PARIS-mediatedtranscriptional repression and specificity but the repression of PARISrelied on the genomic context rather than simple IRS motif. In addition,under certain contexts it appeared as though PARIS could act atranscriptional activator. Consistent with this notion were observationsthat PARIS bound the PEPCK or G6Pase endogenous promoters via ChIP andit had no effect on G6Pase promoter-reporter activity, but enhancedPEPCK promoter-reporter activity. DNA response elements could functionas allosteric effectors that determined the transcriptional activity ofregulators, explaining that regulators may activate transcription in thecontext of one gene, yet repress transcription in another. Indeed, theIRS motifs in the PGC-1α promoter were organized differently then theIRS motifs of the PEPCK and G6Pase promoters, which may, in partaccounted for the ability of PARIS to act as both as a repressor andactivator. Other IRS/PLM responsive genes were likely regulated byPARIS.

Dopaminergic Degeneration in Conditional Parkin KO Mice

Germline deletion of parkin using a variety of approaches created parkinKO mice with minimal phenotypes and no loss of DA neurons. A number ofreasons have been put forward regarding the lack of overt degenerationof DA neurons in genetic animal models of PD including compensatorymechanisms. Similar compensatory mechanisms likely to occur in PD, andcompensation accounts, in part, for the age-dependence of PD. Theultimate failure of these compensatory mechanisms contributes toneurodegeneration.

Since, germline parkin exon 7 KO mice did not have degeneration of DAneurons, conditional parkin exon 7 KO mice were generated and deletedparkin from adult mice to avoid developmental compensation. Similar togermline deletion of parkin, embryonic deletion ofglial-cell-line-derived neurotrophic factor (GDNF) had no deleteriouseffects on DA neurons. However, conditional KO of GDNF in adult animalsusing a tamoxifen sensitive Cre-Lox recombination unmasked the “truephysiologic action of GDNF” and led to mice with profound degenerationof catecholaminergic neurons, indicating that developmental compensationcould be overcome by deleting genes in the DA system in adulthood.Similar to the adult GDNF deleted mice; progressive loss of DA neuronswas observed when parkin was deleted in adult mice. PARIS levelsincreased in parkin conditional KO mice similar to levels in AR-PD brainand in sporadic PD SN. Accompanying the upregulation of PARIS wasdownregulation of PGC-1α and NRF in conditional KO mice similar to thedownregulation in sporadic PD striatum and SN. Consistent with thenotion that compensation occurred in the germline parkin KO mice was theobservation that PARIS was only modestly elevated and there was noalteration in the levels of PGC-1α and NRF and there was no loss of DAneurons.

Parkin, PARIS, PGC-1α and Neurodegeneration

It was not certain that PARIS was the sole substrate or mechanism thatcontributed to DA neuron degeneration following parkin inactivation.Studies suggested that PINK1 in a mitochondrial membranepotential-dependent manner signaled and recruited parkin from thecytoplasm to the mitochondria to initiate degradation of damagedmitochondria through autophagy (mitophagy). Since PGC-1α and NRF-1 weremajor transcriptional regulators of mitochondrial biogenesis, it wasconceivable that when parkin decreased the number of mitochondriathrough mitophagy in response to mitochondrial damage, it was counterbalanced by downregulation of PARIS levels as a homeostatic mechanism toincrease mitochondrial size and number through regulation of PGC-1α andNRF-1 levels. The contribution of other parkin substrates that wereregulated by the UPS and accumulate in AR-PD, sporadic PD and models ofparkin inactivation, such as the aminoacyl-tRNA synthetase interactingmultifunctional protein type 2 (AIMP2) also known as p38/JTV-1 and farupstream element-binding protein 1 (FBP-1), as well as, others were notknown. PARIS upregulation was necessary and sufficient to cause DAneuron degeneration in models of parkin inactivation and thatmaintaining PGC-1α function was beneficial. PGC-1α had been implicatedin other models of PD. Moreover, PGC-1α-responsive genes wereunderexpressed in microdissected dopaminergic neurons of PD suggestingthat the alteration of PGC-1α was a cause of PD pathogenesis, not theconsequence.

A model was developed that accumulated PARIS in the setting of parkin'sinactivation repressed PGC-1α expression leading to neurodegeneration.Consistent with this model were the observations that the reduction inPGC-1α levels and neurodegeneration were substantially reduced byknocking down PARIS levels in the setting of parkin inactivation. SincePARIS was likely regulated other genes, the Parkin-PARIS-PGC-1α was onepotential contributory mechanism to PD pathogenesis. Thisparkin-PARIS-PGC-1α neurodegenerative pathway ultimately resulted in theselective vulnerability of dopamine neurons and accounted, in part, forthe neurodegeneration in PD (FIG. 2). The results suggested that parkininactivation acting through PARIS and downregulation of PGC-1αcontributed to the pathogenesis of PD.

EXPERIMENTS Experiment Methods

Yeast Two-Hybrid Screening

Saccharomyces cerevisiae MaV203 was transformed with pDBLeu-R1-parkin,and 3×10⁶ stable transformants were further transformed with 15 μg ofpPC86 human brain cDNA library (Life Tech/Gibco). Transformants wereselected and confirmed according to the manufacturer's instructions aspreviously described (Zhang et al., 2000).

Antibodies

Polyclonal PARIS antibodies were generated. Primary antibodies usedinclude the following: goat anti-PGC-1α (K-15, Santa CruzBiotechnology), mouse anti-PGC-1α (4C1.3, Calbiochem), goat anti-NRF1(A-19, Santa Cruz Biotechnology), rabbit anti-NRF1 (ab34682, Abcam),mouse anti-parkin (Park8, Cell Signaling), rabbit anti-TH (NovusBiologicals), rabbit anti-glutamate decarboxylase (GAD) 65&67(Millipore), rabbit anti-GFP (ab661, Abcam), mouse anti-GFP (ab1218,Abcam); Secondary antibodies used include Biotin-SP-conjugated goatanti-rabbit (Jackson ImmunoResearch lab), donkey anti-goat-Cy3, donkeyanti-rabbit-Cy2/Cy3, donkey anti-mouse-Cy2 for immunostaining.

Plasmid Constructions

Full-length parkin, and deletion mutants, Q311 X, R42P, R275W, G430D andC431F parkin, HA-ubiquitin, PARIS, PARIS deletion and point mutations,GST-PARIS, ZNF398 vector were constructed. Construct integrity wasverified by sequencing. Lentiviral pLV-PGC-1α plasmid was kindlyprovided by Dr. Dimitri Krainc (Massachusetts General Hospital, HarvardMedical School, Charlestown, USA), MYC-tagged XIAP was generously givenfrom Dr. Kenny K. K. Chung (Hong Kong University of Science andTechnology, Clear Water Bay, Hong Kong).

Cell Culture and Transfection

Human neuroblastoma SH-SY5Y cells (ATCC, Manassas, Va.) were grown inDMEM containing 10% FBS and antibiotics in a humidified 5% CO₂/95% airatmosphere at 37° C. For transient transfection, cells were transfectedwith indicated amounts of target vector using Lipofectamine Plus(Invitrogen), according to manufacturer's instructions. Forco-immunoprecipitation from cell cultures, SH-SY5Y cells weretransfected with 2 μg of each plasmid, unless otherwise indicated. Forthe ubiquitination assay, SH-SY5Y cells were transiently transfectedwith 2 μg of pRK5-Myc-tagged parkin, Myc-tagged parkin (C431F, G430D,R275W, and Q311 X) pCMV-FLAG-PARIS, and 2 μg of pMT123-HA-ubiquitinplasmids for 48 hours. For the luciferase assay SH-SY5Y cells weretransiently transfected with pCMV-empty vector or pCMV-FLAG-PARIS witheither wild type or Q311X parkin, or pGL3-Basic, pGL3-PGC-1αpromoter-Luciferase, or pGL3-PGC-1α promoter deletion mutant (a giftfrom Akyoshi Fukamizu, University of Tsukuba, Japan) (Daitoku et al.,2003) for firefly Luciferase assay and 0.1 μg pRL-TK vector (Promega)for Renilla luciferase control.

Immunocytochemistry and Immunoblot Analysis

Immunocytochemistry and immunoblot analysis was performed.

In Vitro Interaction and Ubiquitination Assays

GST-PARIS and His-Parkin were used in in vitro interaction andubiquitination assays.

CAST, EMSA, ChIP, qRT-PCR Assays

A previously published protocol with modification for CAST ((CyclicAmplification and Selection of Targets) (Voz et al., 2000) was used.GST, GST-PARIS and GST-0571A-PARIS were used for electrophoreticmobility shift assays (EMSA). Chromatin immunoprecipitation was carriedout according to the manufacturer's instruction. Primers used forreal-time pRT-PCR are listed in Table 4.

Conditional Parkin Knockout

A lentiviral vector expressing GFP fused Cre recombinase (Lenti-GFPCre)was stereotaxically introduced into exon 7 floxed parkin mice(parkin^(Flx/Flx)) to generate Cre-flox conditional model of parkinknock out. Furthermore lentiviral shRNA-PARIS was co-administrated alongwith Lenti-GFPCre to demonstrate whether the changes in PGC-1α and NRF-1are due to PARIS.

Antibodies

A peptide containing amino acids 572 to 590 [GKSFIRKDHLRKHQRNHAA] (SEQID NO. 1) was generated from the C-terminal region and cross-linked tokeyhole limpet hemocyanin to generate a peptide antigen of PARIS. Theconjugated peptide was used to immunize a New Zealand white rabbit (JH786-789) (Cocalico Biologicals). Antisera were purified by affinitychromatography using the same peptide immobilized on SulfoLink gelmatrix (Pierce), according to manufacturer's protocol. The quality ofantibody against PARIS is shown in FIGS. 3-8.

Database Searching

Full-length PARIS sequence of human (Accession: Q6NUN9) [SEQ ID NO. 2],mouse (Accession: XP_(—)909399) [SEQ ID NO. 3] and rat (Accession:XP_(—)231752) [SEQ ID NO. 4] were obtained from NCBI(http://www.ncbi.nlm.nih.gov/) and entered as a query for multiplealignment search (ClustalW, http://www.ebi.ac.uk/Tools/clustalw2/).Jalview (http://www.jalview.org/) was used for color-coding editing ofmultiple alignments. Phylogenetic comparison was performed in ClustalW.

Northern Blot Analysis

A multiple human tissue Northern blot (Clontech) was hybridized usingthe PARIS cDNA probe (Y2H clone) labeled with DIG-DNA Labeling Mixture(Roche Diagnostics). Hybridization and washing were performed accordingto manufacturer's instructions, and the PARIS mRNA was detected using aDIG Luminescent Detection Kit (Roche Diagnostics). Levels of PARIS mRNAwere normalized to GAPDH.

Plasmid Constructions

Full-length, deletion mutants, Q311 X, R42P, R275W, G430D and C431Fparkin were cloned into pRK5-MYC vector, and full-length HA-ubiquitinwas cloned into pRK5-HA vector as described previously (Chung et al.,2001; Zhang et al., 2000). Full-length parkin and truncations encodingamino acids 1-198 (UBL-SH) and 220-465 (R1-IBR-R2) were cloned intopRK5-HA vector between SalI and NotI sites. Full-length PARIS cDNA(IMAGE: 30347892; Open Biosystems) was cloned into the mammalianexpression vector pCMV-Tag2A (Stratagene) between EcoRI and XhoI sites.PARIS cDNA sequences encoding amino acids 1-322, 322-644, and 1-164 werecloned into pCMV-Tag2A vector between EcoRI and XhoI sites to generatePARIS truncations. For the ZNF398 vector, its cDNA was amplified withEcoRI or XhoI site-flanked primers and inserted into pCMV-Tag2A vector.The sequences were confirmed by automated DNA sequencing.

Site-Directed Mutagenesis

The expression plasmid, pEGFP-PARIS mutants was generated using aQuikChange site-directed mutagenesis kit (Stratagene). The sequenceswere confirmed by automated DNA sequencing. Primers used are listed onTable 6.

Purification of GST-PARIS Recombinant Proteins

Full-length PARIS (a.a. 1-644) and PARIS zinc finger domain (ZFD) (a.a.453-589) cDNA were PCR-amplified from pCMV-Tag2A-PARIS plasmid andcloned into pGEX-6P-1 vector (GE Healthcare). The sequences wereconfirmed by automated DNA sequencing. The plasmids were transformed toBL21 pLys, which were then grown in the presence of 0.1 mM IPTG for 4hours at 30° C. Cells were lysed by sonication in a THE buffer (10 mMTris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA) containing 0.1% Triton X-100and protease inhibitors and finally centrifuged at 14,000 rpm for 30 minat 4° C. After centrifugation, the supernatant was recovered, and theGST-PARIS and GST-PARIS-ZFD were purified with glutathione Sepharose 4B(GE Healthcare). The GST protein was also prepared as a control. Thepurity and quantity of GST-PARIS and GST-PARIS-ZFD were analyzed bySDS-PAGE with a well-defined BSA concentration standard.

In Vitro Interaction Assay

0.2 μg of GST or GST-PARIS was incubated for 1 hour at 4° C. with 20 μlof glutathione-sepharose beads, respectively for in vitroprotein-protein interaction assays. After washing, GST or GST-PARISconjugated beads were resuspended in 100 μl binding buffer (20 mMTris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS) includingthe protease inhibitor cocktail (Roche), and incubated for 2 hrs at 4°C. with the His-Parkin (Boston Biochem). After extensive washing,retained proteins were eluted by boiling in SDS protein loading bufferand analyzed by immunoblotting using anti-GST and anti-PARIS antibodies.

In Vitro Ubiquitination Assay

GST-PARIS, E1 (50 nM) and different E2s (UbcHs) (50 nM) were incubatedwith His-tagged parkin (100 nM) in presence or absence of ChIP at 37° C.in reaction buffer containing 50 mM Tris-Cl, pH 7.5, 2.5 mM MgCl₂, 2 mMDTT, 2 mM ATP. For reducing conditions, samples were treated with SDSsample buffer and the boiled supernatants were separated by 8-16%gradient SDS-PAGE. Both polymerized ubiquitin chains and ubiquitinatedproteins were detected by immunoblot with anti-ubiquitin (DAKO),anti-ubiquitin, K48-specific (Apu2, Millipore), anti-ubiquitin,K63-specific (Apu3, Millipore), anti-ubiquitin, K63-specific (HWA4C4,Millipore), and anti-PARIS antibody. Recombinant E1, UbcHs and ubiquitinwere purchased from Calbiochem. GST-PARIS was purified from Escherichiacoli strain, BL21 pLys (Stratagene).

Co-Immunoprecipitation

SH-SY5Y cells were transfected with 2 μg of each plasmid, unlessotherwise indicated, for co-immunoprecipitation from cell cultures.After 48 hours, cells were washed with cold PBS and harvested inimmunoprecipitation buffer (1% Triton X-100, 2 μg/ml aprotinin, and 100μg/ml PMSF in PBS). The lysate was then rotated at 4° C. for 1 hour,followed by centrifugation at 14,000 rpm for 20 min. The supernatantswere then combined with 50 μl of protein G Sepharose (AmershamBiosciences) preincubated with antibodies against FLAG or MYC (Sigma;Roche, Indianapolis, Ind.), followed by rotating at 4° C. for 2 hours.The protein G Sepharose was pelleted and washed three times usingimmunoprecipitation buffer or buffer with additional 500 mM NaCl,followed by three washes with PBS. The precipitates were resolved onSDS-PAGE gel and subjected to immunoblot analysis. Immunoblot signalswere visualized with chemiluminescence (Pierce, Rockford, Ill.). Forco-immunoprecipitation of endogenous proteins from mouse brain, adulthuman brain was homogenized in lysis buffer [10 mM Tris-HCl, pH 7.4, 150mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 10 mM Na-β-glycerophosphate,Phosphatase Inhibitor Cocktail I and II (Sigma), and Complete ProteaseInhibitor Mixture (Roche)], using a Diax 900 homogenizer (Heidolph). Thetissue homogenate was incubated on ice for 30 min and mixed twice forcomplete lysis. The samples were then centrifuged at 52,000 rpm at 4° C.for 20 min. The supernatant was used for immunoprecipitation with one ofthe following antibodies: mouse or rabbit IgG (mlgG or rlgG),anti-PARIS, or anti-parkin. Immunoprecipitates were separated bySDS-PAGE and subjected to immunoblot analysis with an anti-PARIS oranti-parkin antibody. Immunoblot signals were visualized withchemiluminescence. For mapping of the binding region between parkin andPARIS, MYC- or HA-tagged parkin deletion constructs were transfectedwith full-length FLAG-PARIS or FLAG-tagged PARIS deletion fragments wereco-transfected with full-length parkin. Transfections andco-immunoprecipitation was performed as described above.

Cellular Ubiquitination Assay

SH-SY5Y cells were transiently transfected with 2 μg of pRK5-Myc-taggedparkin or Myc-tagged parkin (C431F, G430D, R275W, and Q311 X),pCMV-FLAG-PARIS, and 2 μg of pMT123-HA-ubiquitin plasmids for 48 hoursfor the ubiquitination assay. Total cell lysates were prepared byharvesting the cells after washing with PBS, followed by solubilizingthe pellets in 200 μl of 2% SDS, followed by sonication. The lysateswere then rotated at 4° C. for 1 hour, diluted to 1 ml with PBS, andthen boiled and sonicated. The samples were used as input and forimmunoprecipitation. Immunoprecipitation was performed with an antibodyagainst FLAG. The precipitates were subjected to immunoblotting withanti-HA or anti-FLAG antibodies.

Immunocytochemistry

About 5×10⁴ SH-SY5Y cells or rat cortical neurons were seeded ontopolylysine-coated sterile glass cover slips in a 24-well culture plate.After attachment, cells were washed once with PBS and fixed in 3%paraformaldehyde (w/v) for 20 min. The fixed cells were washed threetimes with PBS before permeabilization in 0.2% (v/v) Triton X-100 in PBSfor 5 min. Blocking was then carried out with 5% goat serum in PBS for 1hour. This was followed by incubation in primary antibodies for 1 hourat 25° C. and secondary antibodies for another hour at 25° C.Immunofluorescent images were acquired on a Carl Zeiss confocalmicroscope. For immunohistochemistry with mouse brain, animals wereperfused with PBS followed by 4% paraformaldehyde. Brains werepost-fixed with 4% paraformaldehyde, cryoprotected in 30% sucrose.Sagittal or coronal sections were cut throughout the whole brain andsections were reacted with rabbit polyclonal anti-PARIS and visualizedwith biotinylated goat anti-rabbit IgG, followed bystreptavidin-conjugated horseradish peroxidase (HRP) (Vectastain ABCkit, Vector Laboratories). Positive immunostaining was visualized with3,3′-diaminobenzidine (DAB, Sigma) after reaction with hydrogen peroxide(DAB kit, Vector Laboratories). Stained sections were mounted ontoslides and analyzed by Stereo Investigator software (MicroBrightfield).

Preparation of Tissues for Immunoblot

The tissues including nine different organs, eight different brainregions from C57/BL6 mouse, human brain, and mouse brain werehomogenized in lysis buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mMEDTA, 0.5% Nonidet P-40, 10 mM Na-β-glycerophosphate, PhosphateInhibitor Cocktail I and II (Sigma), and Complete Protease InhibitorMixture (Roche)], using a Diax 900 homogenizer. After homogenization,samples were rotated at 4° C. for 30 min for complete lysis, then thehomogenate was centrifuged at 52,000 rpm for 20 min, and the resultingfractions were collected. Protein levels were quantified using the BCAkit (Pierce) with BSA standards and analyzed by immunoblot.Immunoblotting was performed with an antibody of interest and wasperformed with chemiluminescence (Pierce). The densitometric analyses ofthe bands were performed using ImageJ (NIH,http://rsb.info.nih.gov/ij/). Data are expressed as mean±S.E.M. Theresults were evaluated for statistical significance by applying theunpaired two-tailed Student's t-test or Student-Newman-Keuls.Differences were considered significant when p<0.05.

Luciferase Assay

SH-SY5Y cells were transiently transfected with pCMV-empty vector orpCMV-FLAG-PARIS with either wild type or Q311X parkin. In addition, eachwell was co-transfected with pGL3-Basic, pGL3-PGC-1αpromoter-Luciferase, -pGL3-PGC-1α promoter deletion mutant (a gift fromAkyoshi Fukamizu, University of Tsukuba, Japan) (Daitoku et al., 2003),pGL3 Hygro-rat PEPCK, pGL3 MOD-mouse G6Pase (kindly provided by RichardO'Brien, Vanderbilt University, USA) (Boustead et al., 2003) for fireflyLuciferase assay and 0.1 μg pRL-TK vector (Promega) for Renillaluciferase control. Cells were harvested 48 hours post-transfection andlysates were assayed sequentially for firefly and Renilla luciferases,using the Dual-Luciferase Reporter Assay System (Promega) with aMonolight 3010 luminometer (Analytical luminescence Lab), according tothe manufacturer's instructions. Firefly luciferase readings werenormalized to Renilla readings.

CAST (Cyclic Amplification and Selection of Targets)

The published protocol with modification by Voz et al. (2000) was used.Oligonucleotides containing random 26 nucleotides(CAST26-CTGTCGGAATTCGCTGACGT-(N)26-CGTCTTATCGGATCCTACGT) [SEQ ID NO. 5]were used for generation of random double-strand oligomers for the firstround of CAST, 400 pmol of CAST26 were applied into 100 μl of PCR buffercontaining 1 μmol of CAST-C(ACGTAGGATCCGATAAGACG) [SEQ ID NO. 6], 200 μMdNTP, and 10 units of Taq (Invitrogen) and incubated as follows: 5 minat 94° C., 20 min at 65° C., and 20 min at 72° C. Fifty μl of randomdouble-strand oligomers were subjected to pull-down with GST-ZF-PARIS(322-644 a.a.) bound to Glutathione Sepharose beads in mixture of 50 μgof BSA and 50 μg of poly polydeoscyinosinic-deoscycytidylic acid (Sigma)in 500 μl of binding buffer containing 10 mM Tris (pH 7.5), 200 mM NaCl,10% glycerol, 50 mM ZnCl₂, 1 mM MgCl₂, and 1 mM DTT. Theoligonucleotides were extracted from the beads by applying 100 μl ofdistilled H₂O, followed by phenol extraction and ethanol precipitation.An elute was used for the subsequent PCR in the presence of 200 pmol ofeach primer CAST-N(CTGTCGGAATTCGCTGACG) [SEQ ID NO. 7] and CAST-C with25 cycles of 1 min at 94° C., 1 min at 65° C., and 1 min at 72° C. Afterfour rounds of selection were done, an additional three rounds ofselection were performed by EMSA. An eluate from 4th round CAST wasamplified with 1 μl of [α-³²P]-dCTP (GE Healthcare), incubated withGST-ZF-PARIS, loaded on PAGE. DNA was extracted from the shifted band onEMSA and subsequently used for a second round of selection performed asdescribed above. Following a total of seven selections, oligomers werecloned into the pGEM-T Easy vector according to the manufacturer'sprotocol (Promega). Twenty-four independent clones were sequenced and 19clones were identifiable. Among 19 clones, 3 clones were duplicated andthe final 16 clones were aligned with MACAW software (NCBI,http://iubio.bio.indiana.edu/soft/molbio/ncbi/old/macaw/).

Cyclic amplification and selection of targets (CASTing) is essentiallyidentical to the selected and amplified (protein) binding siteoligonucleotide (SAAB) and target detection assay (TDA) procedures; aprocedure for identification of consensus sequences of DNA to which aprotein, e.g. a transcription factor, may bind. A random polynucleotidesequence is synthesized flanked by two defined sequences that will serveas templates for PCR primers; the polynucleotides are exposed to theDNA-binding protein, any complex that is formed is separated from theunliganded polynucleotides (e.g. by gel shift assay, affinitychromatography, filter binding) and the polynucleotide of the complex isisolated and amplified by PCR; repeated recycling through the sequenceof ligand formation, selection and amplification results in apreparation that is sufficiently pure to be cloned into bacteria forlarger-scale production. A variant is systematic evolution of ligands byexponential enrichment (SELEX) for identification of RNA sequences,which begins with a mixture of polyribonucleotides and in each cycleproduces DNA from the selected RNA-protein complex using reversetranscriptase, amplifies it by PCR, and then produces new RNAtranscripts for the next round of selection.

EMSA

GST, GST-PARIS, and GST-0571A-PARIS were prepared as described above.The different probes for the WT-IRS and Mutant-IRS with mutations in theconsensus sequence (underline) were synthesized as followed:

[SEQ ID NO. 8] IRS1-WT: ⁹⁸⁶AGTGTGTTGGTATTTTTCCCTCAGTTC⁹⁶⁰ [SEQ ID NO. 9]IRS1-MT: ⁹⁸⁶AGTGTGTTGGTATTGTTCCCTCAGTTC⁹⁶⁰ [SEQ ID NO. 10]IRS2-WT: ⁵⁹⁶ACATACAGGCTATTTTGTTGATTAAAC⁵⁷⁰ [SEQ ID NO. 11]IRS2-MT: ⁵⁹⁶ACATACAGGCTATTGTGTTGATTAAAC⁵⁷⁰ [SEQ ID NO. 12]IRS3-WT: ³⁶⁴GCCACTTGCTTGTTTTGGAAGGAAAAT³³⁸ [SEQ ID NO. 13]IRS3-MT: ³⁶⁴GCCACTTGCTTGTTGTGGAAGGAAAAT³³⁸

The complementary probes were annealed in buffer consisting of 100 mMNaCl, 10 mM Tris-Cl (pH 8.0), and 1 mM EDTA, subsequently end-labeledwith [γ-³²P]ATP (GE Healthcare) in present of T4 polynucleotide kinase(Promega), and finally purified with the QIAquick Nucleotide removal kit(Qiagen). Probe-protein binding reactions were performed for 10 min atroom temperature in 25 μl of binding buffer consisting of 10 mM Tris (pH7.9), 4% glycerol, 100 mM KCl, 50 mM ZnCl₂, 1 mM DTT, 1 mgpolydeoxyadenylic acid-polythymidylic acid (Sigma), and 10 μg of BSA.Probe-protein complexes were analyzed on 5% nondenaturing polyacrylamidegels and electrophoresis was carried out at 4° C.

Chromatin Immunoprecipitation (ChIP)

Chromatin immunoprecipitation was carried out according tomanufacturer's instruction (Millipore) with modification. Briefly,powderized brain (mouse and human) was suspended in 1% formaldehyde inPBS for 20 min at room temperature and SH-SY5Y cells were fixed with 1%formaldehyde for 10 min at 37° C. Glycerol quenched samples were lysedin 1 ml of SDS buffer containing protease inhibitors. The lysates wereincubated for 10 min on ice and sonicated to shear DNA. The samples werecentrifuged at 10,000×g at 4° C. for 10 min and supernatant was taken.Pre-cleared samples were incubated with either PARIS or rabbit IgG(rlgG)-agarose bead followed by a number of washes. Elutes weresubjected to reverse cross-linking and DNA was recovered byphenol-chloroform-ethanol purification. PCR was performed using templateDNA and the following primers:

hPGC-1a promoter (forward, 5′-ACATACAGGCTATTTTGTTGATTAAAC-3′[SEQ ID NO. 14]; reverse, 5′-ATTTTCCTTCCAAAACAAGCAAGTGGC-3′[SEQ ID NO. 15]), hG6Pase promoter(forward, 5′-GTAGACTCTGTCCTGTGTCTCTGGCCTG-3′ [SEQ ID NO. 16]; reverse,5′-GGTCAACCCAGCCCTGATCTTTGGACTC-3′ [SEQ ID NO. 17]), hPEPCK promoter(forward, 5′-GACTGTGACCTTTGACTATGGGGTGACATC-3′ [SEQ ID NO. 18]; reverse,5′-CTGGATCACGGCCAGGGTCAGTTATGC-3′ [SEQ ID NO. 19]), hGAPDH promoter(forward, 5′-TACTAGCGGTTTTACGGGCG-3′ [SEQ ID NO. 20]; reverse,5′-TCGAACAGGAGGAGCAGAGAGCGA-3′ [SEQ ID NO. 21]), mPGC-1 a promoter(forward, 5′-CAAAGCTGGCTTCAGTCACA-3′ [SEQ ID NO. 22]; reverse,5′-TTGCTGCACAAACTCCTGAC-3′ [SEQ ID NO. 23]), and mGAPDH promoter(forward 5′-TGGGTGGAGTGTCCTTTATCC-3′ [SEQ ID NO. 24]; reverse5′-TATGCCCGAGGACAATAAGG-3′ [SEQ ID NO. 25]).Real-Time Quantitative RT-PCR (qRT-PCR)

Total RNA was extracted with Trizol reagent (Invitrogen), and cDNA wassynthesized from total RNA (1.5 μg) using a First-strand cDNA synthesiskit (Invitrogen). Aliquots of cDNA were used as templates for real-timeqRT-PCR procedure. Relative quantities of mRNA expression were analyzedusing real-time PCR (Applied Biosystems ABI Prism 7700 SequenceDetection System, Applied Biosystems). The SYBR greenER reagent(Invitrogen) was used according to the manufacturer's instruction. Formicrodissected specimens, RNA was extracted with proteinase K/acidphenol method (Khodosevich et al., 2007). To eliminate DNA, dissolvedRNA was treated with DNase I (RNase free, Stratagene) for 15 min at 37°C. and purified by RNeasy kit (Qiagen). RNA was directly used forqRT-PCR according to the manufacturer's instruction (QuantiTect SYBRGreen RT-PCR kit, Qiagen). The primer sequences are listed in Table 4.

AAV1-Plasmid Construction and Generation of AAV1 Virus

cDNAs for PARIS and parkin were sub-cloned into an AAV1 expressionplasmid (AAV/CBA-WPRE-bGHpA) under the control of a CBA (chickenbeta-actin) promoter and containing WPRE (woodchuck hepatitis viruspost-transcriptional-regulatory element), and bovine growth hormonepolyadenylation signal flanked by AAV2 inverted terminal repeats (ITRs).dGFP (destabilized GFP) was cloned into the same AAV expression vectorbackbone and was used as control vector. High-titer AAV virus generationand purification were performed as described in detail elsewhere (Duringet al., 2003).

Lentiviral shRNA Constructs

MISSION short hairpin RNA (shRNA) plasmids encoding small interferingRNAs (siRNAs) targeting parkin or PARIS were purchased from Sigma (StLouis, Mo.). TRCN0000000285 and TRCN0000000283 vectors successfullyknockdown human parkin. Three plasmids (TRCN0000156627 TRCN0000157534and TRCN0000157931) were effective in knocking down PARIS expression. Asa control, shRNA-dsRed co-expressing GFP and short hairpin sequence(AGTTCCAGTACGGCTCCAA) [SEQ ID NO. 26] under the control of the EF1α andhuman U6 promoter was used. For knockdown human parkin or PARIS inSH-SY5Y cells, two lentiviral vectors were combined and theTRCN0000157931 lentiviral vector was used to knockdown mouse PARIS invivo.

Stereological Assessment

Experimental procedures were followed for stereotaxic injection of AAV1overexpressing GFP, PARIS or parkin and lentivirus overexpressingPGC-1α, GFP or GFPCre. Six-week-old male C57BL mice (Charles RiverLaboratories, Inc) or 6˜8 week old parkin^(flx/flx) mice wereanesthetized with pentobarbital (60 mg/kg). An injection cannula (26.5gauge) was applied stereotaxically into the substantia nigra(anteroposterior, −3.0 mm from bregma; mediolateral, 1.2 mm;dorsoventral, 4.3 mm). The infusion was performed at a rate of 0.2μl/min and wound healing and recovery were monitored after the injectionwas done. Four weeks, 3 months and 10 months after injection, animalswere anesthetized and perfused with PBS followed by 4% paraformaldehyde.Brains were post-fixed with 4% paraformaldehyde, cryoprotected in 30%sucrose, and processed for immunohistochemistry. Forty-μm coronalsections were cut throughout the brain including substantia nigra andevery 4^(th) section was utilized for analysis. For tyrosine hydroxylase(TH) or glutamate decarboxylase 65/67 (GAD 65/67), sections were reactedwith a 1:1000 dilution of rabbit polyclonal anti-TH (Novus) oranti-GAD65/67 (Chemicon) and visualized with biotinylated goatanti-rabbit IgG, followed by streptavidin-conjugated horseradishperoxidase (HRP) (Vectastain ABC kit, Vector Laboratories, Burlingame,Calif.). Positive immunostaining was visualized with3,3′-diaminobenzidine (DAB, Sigma) after reaction with hydrogen peroxide(DAB kit, Vector Laboratories). Stained sections were mounted ontoslides and counterstained with thionin for NissI substance. Totalnumbers of TH-, and NissI-stained neurons in substantia nigra parscompacta were counted using the Optical Fractionator probe of StereoInvestigator software (MicroBrightfield, Williston, Vt.). For NissIcounting, a cell was defined as a bright blue-stained neuronal perikaryawith a nucleolus. NissI positive counts were restricted to NissI+/TH+neurons along with large NissI+ neurons with dopaminergic-likemorphology, that contain little or no TH immunostaining.

Laser Capture Microdissection (LCM)

Approximately 6 week old parkin^(flx/flx) mice injected with eitherlentiviral GFP (n=3) or lentiviral GFPCre (n=3) were transcardiallyperfused by autoclaved 1×PBS for 3 min (10 ml/min), 2% paraformaldehyde(resolved in autoclaved PBS) for 5 min (10 ml/min), and 20% sucrose for5 min (10 ml/min). The brains were rapidly removed and frozen on dryice. In order to preserve fluorescence and RNA integrity, an RNaseinhibitor and autoclaved PBS were used during all staining procedures.Fifteen micron-thick coronal sections of the midbrain on superfrostglass slide were incubated with blocking solution for 30 min and rinsedwith 1×PBS followed by incubation with rabbit anti-TH (1:50) and mouseanti-GFP (1:50) for 3-4 hours. Rinsed sections were incubated withCy3-conjugated anti-rabbit (1:25) and Cy2-conjugated anti-mouse (1:25)for 1 hour. Sections were rinsed with 1×PBS three times and were washedonce again with DEPC-treated water. Double, TH and GFP, positive neuronswere obtained by LCM (P.A.L.M., Microlaser Technologies). Microdissectedcells were directly used for RNA extraction.

Statistics

Quantitative data is presented as the mean±S.E.M. Statisticalsignificance was either assessed via an unpaired two-tailed Student'st-test or an ANOVA test with Student-Newman-Keuls post-hoc analysis.Assessments were considered significant with a p<0.05.

Experiment 1 PARIS was a KRAB and C2H2 Zinc Finger Protein

PARIS was identified by yeast two-hybrid screening using parkin as bait.Human PARIS (ZNF746) was a 644 amino acid protein that contained aKruppel-associated box (KRAB) at its N-terminus and a C2HC/C2H2 typezinc finger at its C-terminus (FIG. 9). There was a high degree ofhomology among human, mouse and rat PARIS proteins (FIG. 4) (see Table1). Northern blot and immunoblot analysis revealed that PARIS wasexpressed in all organs examined (FIGS. 5 and 6). PARIS wasdifferentially expressed in the brain with low levels in cerebellum andmidbrain (FIG. 3). Immunohistochemisty revealed that PARIS washeterogeneously distributed throughout the brain and that it waslocalized to neurons, including substantia nigra (SN) pars compacta DAcontaining neurons (FIG. 10). Confocal imaging indicated that PARIS wasco-localized with parkin in primary cortical neuron cultures (FIG. 11)and in SH-SY5Y dopaminergic-like cells (FIG. 7). The polyclonal PARISantibody used in these studies was specific for PARIS since itrecognized a single band on immunoblot from mouse brain (see FIG. 8).

TABLE 1Comparison of full-length PARIS sequence of human [SEQ ID NO. 2],mouse [SEQ ID NO. 3] and rat [SEQ ID NO. 4] MouseMAEAAAAPISPWTMAATIQAMERKIESQAARLLSLEGRTGMAEKKLADCEKTAVEFSNQL 60 RatMAEAAAAPISPWTMAATIQAMERKIESQAARLLSLEGRTGMAEKKLADCEKTAVEFSNQL 60 HumanMAEAVAAPISPWTMAATIQAMERKIESQAARLLSLEGRTGMAEKKLADCEKTAVEFGNQL 60****.***************************************************.*** MouseEGKWAVLGTLLQEYGLLQRRLENVENLLRNRNFWILRLPPGSKGEVPKEWGKLEDWQKEL 120 RatEGKWAVLGTLLQEYGLLQRRLENVENLLRNRNFWILRLPPGSKGEVPKEWGKLEDWQKEL 120 HumanEGKWAVLGTLLQEYGLLQRRLENVENLLRNRNFWILRLPPGSKGESPKEWGKLEDWQKEL 120********************************************* ************** MouseYKHVMRGNYETLVSLDYAISKPEVLSQIEQGKEPCTWRRTGPKVPEVPVDPSPGSGAPVP 180 RatYKHVMRGNYETLVSLDYAISKPEVLSQIEQGKEPCTWRRTGPKVPEVPVDPSPGSGAPVP 180 HumanYKHVMRGNYETLVSLDYAISKPEVLSQIEQGKEPCNWRRPGPKIPDVPVDPSPGSGPPVP 180***********************************.***.***:*:**********.*** MouseAPDLLMQIKQEGELQLQEQQALGVEAWAAGQPDIGEEPWGLSQLDSGAGDISTDATSGVH 240 RatAPDLLMQIKQEGELQLQEQQALGVEAWAAGQPDIGEEPWGLSQLDSGAGDISTDATSGVH 240 HumanAPDLLMQIKQEGELQLQEQQALGVEAWAAGQPDIGEEPWGLSQLDSGAGDISTDATSGVH 240************************************************************ MouseSNFSTTIPPTSWQADLPPHHPSSACSDGTLKLNTAASTEADVKIVIKTEVQEEEVVATPV 300 RatSNFSTTIPPTSWQADLPPHHPSSACSDGTLKLNTAASTEADVKIVIKTEVQEEEVVATPV 300 HumanSNFSTTIPPTSWQTDLPPHHPSSACSDGTLKLNTAASTE-DVKIVIKTEVQEEEVVATPV 299*************:************************* ******************** MouseHPTDLEAHGTLFAPGQATRFFPSPVQEGAWESQGSSFPSQDPVLGLREPTRPERDIGELS 360 RatHPTDLEAHGTLFAPGQATRFFPSPVQEGAWESQGSSFPSQDPVLGLREPTRPERDIGELS 360 HumanHPTDLEAHGTLFGPGQATRFFPSPAQEGAWESQGSSFPSQDPVLGLREPARPERDMGELS 359************.***********.************************:*****:**** MousePAIAQEEAPAGDWLFGGVRWGWNFRCKPPVGLNPRTVPEGLPFSSPDNGEAILDPSQAPR 420 RatPAIAQEEAPAGDWLFGGVRWGWNFRCKPPVSLNPRTVPEGLPFSSPDNGEAILDPSQAPR 420 HumanPAVAQEETPPGDWLFGGVRWGWNFRCKPPVGLNPRTGPEGLPYSSPDNGEAILDPSQAPR 419**:****:*.********************.*****.*****:***************** MousePFNDPCKYPGRTKGFGHKPGLKKHPAAPPGGRPFTCATCGKSFQLQVSLSAHQRSCGLSD 480 RatPFNDPCKYPGRTKGFGHKPGLKKHPAAPPGGRPFTCATCGKSFQLQVSLSAHQRSCGLSD 480 HumanPFNEPCKYPGRTKGFGHKPGLKKHPAAPPGGRPFTCATCGKSFQLQVSLSAHQRSCGAPD 479***:***************************************************** .* MouseGAATGAASTTTGGGGGGSGGGGGSSGGGSSARDSSALRCGECGRCFTRPAHLIRHRMLHT 540 RatGAGTGAASTATGGGGGG--GGGGSSAGGSSARDSSALRCGECGRCFTRPAHLIRHRMLHT 538 HumanGSGPG-----TGGGGSGSGGGGGGSGGGS-ARDGSALRCGECGRCFTRPAHLIRHRMLHT 533*:..*     *****.*  ****.*.*** ***.************************** MouseGERPFPCTECEKRFTERSKLIDHYRTHTGVRPFTCTVCGKSFIRKDHLRKHQRNHPAVAK 600 RatGERPFPCTECEKRFTERSKLIDHYRTHTGVRPFTCTVCGKSFIRKDHLRKHQRNHPAVAK 598 HumanGERPFPCTECEKRFTERSKLIDHYRTHTGVRPFTCTVCGKSFIRKDHLRKHQRNHAAGAK 593*******************************************************.* ** MouseAPAHGQPLPPLPAPPDPFKSPAAKGPMASTDLVTDWTCGLSVLGPSDGGGDL 652 RatAPAHGQPLPPLPAPPDPFKSPAAKGPMASTDLVTDWTCGLSVLGPNDGGGDL 650 HumanTPARGQPLPTPPAPPDPFKSPASKGPLASTDLVTDWTCGLSVLGPTDGG-DM 644:**:*****. ***********:***:******************.*** *:

Experiment 2 Parkin Interacted with PARIS

MYC-tagged parkin and FLAG-tagged PARIS co-immunoprecipitated in SH-SY5Ycells, whereas XIAP, a RING finger ubiquitin E3 ligase and ZNF398, ahighly conserved homologue of PARIS did not interact with PARIS andparkin, respectively (FIG. 12). Recombinant GST-PARIS pulled downrecombinant His-Parkin indicating that PARIS and parkin directlyinteracted (FIG. 13). The familial PD-associated mutations in parkin,C431F, R275W and Q311X bound to PARIS less avidly than WT parkin,whereas G430D mutant parkin bound to PARIS in a manner similar to WTparkin (FIG. 14). PARIS co-immunoprecipitated with parkin and parkinco-immunoprecipitated with PARIS from whole human striatum (FIG. 15) ormouse brain (FIG. 16). This endogenous interaction between parkin andPARIS was not observed in parkin exon 7 KO brain (FIG. 17) confirmingthe specificity of the interaction.

Domain mapping indicated that parkin bound to the C-terminal domain, butnot to the N-terminal domain of PARIS (FIG. 18). To ascertain whichdomain of parkin bound to PARIS, various deletion constructs ofMYC-tagged parkin were utilized. Co-immunoprecipitation experimentsshowed that both the RING1 or RING2 domains were required for parkinbinding to PARIS (FIGS. 19 and 20). Taken together these resultsindicated that parkin interacted with the zinc finger domain of PARISand PARIS bound to either the RING1 or RING2 domain of parkin.

Experiment 3 Parkin Ubiquitinated PARIS and Regulated PARIS Levels

FLAG-tagged PARIS was ubiquitinated by MYC-tagged parkin in SH-SY5Ycells as shown by the substantial HA immunoreactivity in the form of asmear, which was characteristic of polyubiquitinated proteins (lane 3,FIG. 21). Familial linked parkin mutations C431F, G430D and Q311X havesubstantially reduced ubiquitination activity against PARIS, whereasR275W had modest activity (FIG. 21). The reduction in ubiquitination ofPARIS by these mutants was due, in part, to their reduced binding (seeFIG. 14), as well as, reduced E3 ligase activity of these parkinmutants.

FLAG-PARIS was ubiquitinated in the absence of exogenous parkin (lane 2,FIG. 21 and lane 2, FIG. 22). This endogenous ubiquitination wasenhanced with co-expression of WT parkin (lane 3, FIG. 21 and lane 3,FIG. 22). ShRNA-Parkin eliminated the endogenous ubiquitination (lane 4,FIG. 22). To control for potential off-targets effects of shRNA-parkin,a shRNA-resistant WT parkin, but not a shRNA-resistant Q311X parkinmutant restored the ubiquitination of PARIS in the presence ofshRNA-parkin (FIG. 22).

Target sequences for shRNA to PARIS were identified and isolated. Onetarget sequence was the 3′UTR region of PARIS (GCCTCAAAGGAACTTCTGCTT)[SEQ ID NO. 27]. Another target sequence was the coding region of PARIS(GCCATTCAACGAACCCTGTAA) [SEQ ID NO. 28].

In vitro ubiquitin assays showed that parkin ubiquitinates PARIS in thepresence of various E2 enzymes including UbcH5c (FIG. 23). There was noubiquitination signal in the absence of PARIS (FIG. 24) and in theabsence of parkin (FIG. 25) indicating that the ubiquitination signalwas specific for PARIS ubiqutination. ChIP, which acted as an E4 forparkin, enhanced the ubiquitination of PARIS by parkin, but it had noaffect in the absence of parkin indicating that ChIP did notubiquitinate PARIS directly (FIG. 26). OTU1, a K48-linkage specificdeubiquitinating enzyme, successfully hydrolyzed the poly-ubiquitinchain on PARIS (FIGS. 27 and 28) and a K48-specific anti-ubiquitinantibody, but a K63 antibody did not detect the poly-ubiquitin chain onPARIS (FIG. 27), suggesting that endogenous and exogenous parkinubiquitinated PARIS via K48 linkages.

The steady state level of PARIS was regulated by the ubiquitinproteasome system since the level of PARIS increased approximately twoto three fold when SH-SY5Y cells were treated with the proteasomeinhibitor, MG132 (FIG. 29). The levels of PARIS and parkin were tightlyand significantly correlated (R²=0.9985) in co-expression experiments inSH-SY5Y cells in which increased levels of parkin led to decreasedlevels of PARIS (FIG. 30). Cyclohexamide (100 μg/ml) chase experimentsin SH-SY5Y cells transiently expressing GFP-tagged PARIS with or withoutWT parkin or Q311X parkin demonstrated that the decrease in the steadystate levels of PARIS was accelerated in the presence of WT parkincompared with cells transfected with GFP-vector or familial linkedparkin Q311X mutant (FIG. 31). Parkin at a 4 to 1 ratio with PARIS ledto a statistically significant reduction in PARIS that was blocked byMG132, whereas the catalytically inactive Q311X parkin failed to reducePARIS levels (FIG. 32). ShRNA-Parkin led to a significant increase inthe level of PARIS, which was reduced by expression of a shRNA-resistantWT parkin (FIG. 33). These results taken together suggested that parkincontrols the levels of PARIS via the ubiquitin proteasome system.

Experiment 4 PARIS Accumulated in the Absence of Parkin Activity

PARIS levels were increased two fold in the cingulate cortex of AR-PDpatients, which lacked functional parkin, versus age-matched controls(FIGS. 34 and 35) (See Table 2). Due to the lack of tissue availability,it was not possible to assess the levels of PARIS in other brain regionsof AR-PD patients. PARIS levels were also increased by more than twofold in both the striatum and the SN of sporadic PD compared to controls(FIGS. 36 and 37) (See Table 3). No alteration in the levels of PARISmRNA was found in PD striatum or PD SN versus control striatum and SNindicating that the increased level of PARIS was not due to atranscriptional effect (see FIGS. 38 and 39) (See Table 4 for qRT-PCRprimers and Table 5 for details on human brain samples). PARIS levelswere not increased in regions of the brain that were relativelyunaffected in PD including the cerebellum and the frontal cortex ofsporadic PD patients compared to controls (FIGS. 36 and 37) suggestingthat the upregulation was primarily within the nigrostriatal pathway.

TABLE 2 Final Diagnosis Age PMD Control Control 61 15 Control 67 6Control 68 10 Control 49 15 AR-PD PD 62 15 PD 65 4 PD 52 18 PD 68 10

AR-PD cingulate cortex used for immunoblot in FIGS. 34 and 35.Abbreviations: PD, Parkinson's disease; PMD, post-mortem delay.

TABLE 3 Final Diagnosis Age Sex Race PMD Control Control 87 F W 7Control 89 M W 8.5 Control 71 M W 16 Control 79 M W 16 PD PD w/dementia76 M W 17 PD w/dementia, 83 M W 5 neurodegeneration, occipital infarctPD, multiple 80 F W 6 infarcts/contusions PD, Neocortical 71 M W 8 PDw/dementia 73 M W 6.5

Human postmortem tissues used for immunoblot in FIGS. 36 and 37, andChIP in FIGS. 40 and 41. Abbreviations: PD, Parkinson's disease; W,white; B, black; PMD, post-mortem delay.

TABLE 4 Target genes Mouse primers (5′-3′) Human primers (5′-3′) PGC-1αF AGCCGTGACCACTGACAACGAG TCCTCACAGAGACACTAGACA (SEQ ID NO. 29)(SEQ ID NO. 31) R GCTGCATGGTTCTGAGTGCTAAG CTGGTGCCAGTAAGAGCTTCT(SEQ ID NO. 30) (SEQ ID NO. 32) PEPCK F CTGCATAACGGTCTGGACTTCCAAGACGGTTATCGTCACCCA (SEQ ID NO. 33) (SEQ ID NO. 35) RCAGCAACTGCCCGTACTCC GAACCTGGCATTGAACGCTT (SEQ ID NO. 34) (SEQ ID NO. 36)G6Pase F CGACTCGCTATCTCCAAGTGA GTGTCCGTGATCGCAGACC (SEQ ID NO. 37)(SEQ ID NO. 39) R GTTGAACCAGTCTCCGACCA GACGAGGTTGAGCCAGTCTC(SEQ ID NO. 38) (SEQ ID NO. 40) IGFBP-1 F ATCAGCCCATCCTGTGGAACGAGCACGGAGATAACTGAGGA (SEQ ID NO. 41) (SEQ ID NO. 43) RTGCAGCTAATCTCTCTAGCACTT GCCTTCGAGCCATCATAGGTA (SEQ ID NO. 42)(SEQ ID NO. 44) APOC3 F TACAGGGCTACATGGAACAAGC CTCCCTTCTCAGCTTCATGC(SEQ ID NO. 45) (SEQ ID NO. 47) or TGCAGGGTTACATGAAGCACG (SEQ ID NO. 48)R CAGGGATCTGAAGTGATTGTCC GTCTGACCTCAGGGTCCAAA (SEQ ID NO. 46)(SEQ ID NO. 49) or CTCCAGTAGTCTTTCAGGGAACT (SEQ ID NO. 50) TAT FTGCTGGATGTTCGCGTCAATA TGTGTCCCCATCTTAGCTGAT (SEQ ID NO. 51)(SEQ ID NO. 53) or TACAGACCCTGAAGTTACCCAG (SEQ ID NO. 54) RCGGCTTCACCTTCATGTTGTC AATGGTACAGGGTCCCAAAATG (SEQ ID NO. 52)(SEQ ID NO. 55) or TAAGAAGCAATCTCCTCCCGA (SEQ ID NO. 56) SOD1 FCCAGTGCAGGACCTCATTTT AGGGCATCATCAATTTCGAGC (SEQ ID NO. 57)(SEQ ID NO. 59 R TTGTTTCTCATGGACCACCA GCCCACCGTGTTTTCTGGA(SEQ ID NO. 58) (SEQ ID NO. 60) SOD2 F CCGAGGAGAAGTACCACGAGTTGGCCAAGGGAGATGTTAC (SEQ ID NO. 61) (SEQ ID NO. 63) RGCTTGATAGCCTCCAGCAAC AGTCACGTTTGATGGCTTCC (SEQ ID NO. 62)(SEQ ID NO. 64) GPX1 F CCGTGCAATCAGTTCGGACA GCACCCTCTCTTCGCCTTC(SEQ ID NO. 65) (SEQ ID NO. 67) R TCACTTCGCACTTCTCAAACAATTCAGGCTCGATGTCAATGGTC (SEQ ID NO. 66) (SEQ ID NO. 68) CAT FAGCGACCAGATGAAGCAGTG CGCAGAAAGCTGATGTCCTGA (SEQ ID NO. 69)(SEQ ID NO. 71) R TCCGCTCTCTGTCAAAGTGTG TCATGTGTGACCTCAAAGTAGC(SEQ ID NO. 70) (SEQ ID NO. 72) NRF1 F GTTGGTACAGGGGCAACAGTCTTACAAGGTGGGGGACAGA (SEQ ID NO. 73) (SEQ ID NO. 75) RTCGTCTGGATGGTCATTTCA GGTGACTGCGCTGTCTGATA (SEQ ID NO. 74)(SEQ ID NO. 76) TFAM F CCAAAAAGACCTCGTTCAGC CCGAGGTGGTTTTCATCTGT(SEQ ID NO. 77) (SEQ ID NO. 79) R CTTCAGCCATCTGCTCTTCCTCCGCCTATAAGCATCTTG (SEQ ID NO. 78) (SEQ ID NO. 80) UCP2 FACTTTCCCTCTGGATACCGC GCATCGGCCTGTATGATTCT (SEQ ID NO. 81)(SEQ ID NO. 83) R ACGGAGGCAAAGCTCATCTG TTGGTATCTCCGACCACCTC(SEQ ID NO. 82) (SEQ ID NO. 84) UCP3 F CTGCACCGCCAGATGAGTTTAGCCTCACTACCCGGATTTT (SEQ ID NO. 85) (SEQ ID NO. 87) RATCATGGCTTGAAATCGGACC CGTCCATAGTCCCGCTGTAT (SEQ ID NO. 86)(SEQ ID NO. 88) ANT1 F GTCTCTGTCCAGGGCATCAT ATGGTCTGGGCGACTGTATC(SEQ ID NO. 89) (SEQ ID NO. 91) R ACGACGAACAGTCTCAAACGTCAAAGGGGTAGGACACCAG (SEQ ID NO. 90) (SEQ ID NO. 92) ATP5B FGAGGGATTACCACCCATCCT GCACGGAAAATACAGCGTTT (SEQ ID NO. 93)(SEQ ID NO. 95) R CATGATTCTGCCCAAGGTCT GCCAGCTTATCAGCTTTTGC(SEQ ID NO. 94) (SEQ ID NO. 96) CYTC F CCAAATCTCCACGGTCTGTTCGGTGATGTTGAGAAAAGGCAAG (SEQ ID NO. 97) (SEQ ID NO. 99) RATCAGGGTACCTCTCCCCAG GTTCTTATTGGCGGCTGTGT (SEQ ID NO. 98)(SEQ ID NO. 100) COX II F ACGAAATCAACAACCCCGTA TTCATGATCACGCCCTCATA(SEQ ID NO. 101) (SEQ ID NO. 103) R GGCAGAACGACTCGGTTATCTAAAGGATGCGTAGGGATGG (SEQ ID NO. 102) (SEQ ID NO. 104) COX IV FACCAAGCGAATGCTGGACAT CCG CGCTCGTTATCATGTG (SEQ ID NO. 105)(SEQ ID NO. 107) R GGCGGAGAAGCCCTGAA CGTTCTTTTCGTAGTCCCACTTG(SEQ ID NO. 106) (SEQ ID NO. 108) GAPDH F AAACCCATCACCATCTTCCAGAAACCCATCACCATCTTCCAG (SEQ ID NO. 109) (SEQ ID NO. 111) RAGGGGCCATCCACAGTCTTCT AGGGGCCATCCACAGTCTTCT (SEQ ID NO. 110)(SEQ ID NO. 112) Parkin F TGGAAAGCTCCGAGTTCAGT CAGCAGTATGGTGCAGCGGA(SEQ ID NO. 113) (SEQ ID NO. 115) R CCTTGTCTGAGGTTGGGTGTTCAAATACGGCACTGCACTC (SEQ ID NO. 114) (SEQ ID NO. 116) PARIS FAGTTGGACTCTGGAGCAGGA GCTGGAATTTCCGGTGTAAACC (SEQ ID NO. 117)(SEQ ID NO. 119) R GCTGCTGTGTTGAGCTTCAG GGGGTCCAAGATGGCCTCT(SEQ ID NO. 118) (SEQ ID NO. 120)

Primers used for real-time qRT-PCR in FIGS. 38, 39 and 42.Abbreviations: PGC-1α, peroxisome proliferators-activated receptor γcoactivator-1α; PEPCK, phosphoenolpyruvate carboxylase; G6Pase,glucose-6-phosphatase; IGFBP-1, insulin-like growth factor bindingprotein 1; APOC3, apolipoprotein C-Ill; TAT, tyrosine aminotransferase;SOD, superoxide dismutase-1; GPX1, Glutathione Peroxidase 1; CAT,Catalase; NRF1, nuclear respiratory factor 1; TFAM, mitochondrialtranscription factor A; UCP, uncoupling protein; ANT1, adeninenucleotide translocator; ATP5B, ATP synthase, H+ transporting,mitochondrial F1 complex; CYTC, cytochrome c; COX, cytochrome C oxidase;F, forward primer; R, reverse primer (5′-3′); N/A, not applicable.

TABLE 5 Final Diagnosis Age Sex Race PMD Tissue Control Control 62 M W14 Str Control 59 M W 12 Str Control 79 M W 16 Str, SN Control 53 M WN/A Str Control 73 F W 9 SN Control 85 M B 6 SN PD PD, CVD 85 F W 9 Str,SN PD, W/D 60 M W 16 Str PD, W/D, ND, OI 83 M W 5 Str, SN PD, W/D 84 F W11 Str, SN PD, W/D 71 M W 24 SN

Human postmortem tissues used for immunoblot and real time qRT-PCRanalysis in FIGS. 38, 39 and 43-47 and ChIP analysis in FIG. 48.Abbreviations: CVD, cerebrovascular disease; W/D, with dementia; ND,neurodegeneration; OI, occipital infarction; W, white; B, black; PMD,post-mortem delay; N/A, not available; Str, striatum; SN, substantialnigra.

Next, PARIS was evaluated to determine if it was increased in germlineparkin exon 7 KO mice. PARIS was modestly upregulated by approximately48% in the striatum and by 63% in ventral midbrain of germline parkinexon 7 KO mice compared to age matched WT controls, whereas the levelsof PARIS in the cortex was not changed (FIGS. 49 and 50). Theupregulation of PARIS was not due to a transcriptional effect sincethere was no difference in the level of PARIS mRNA in germline parkin KOventral midbrain and striatum versus WT ventral midbrain and striatum(FIG. 51).

Since germline parkin exon 7 KO mice lack parkin from the point ofconception, it was possible that compensatory mechanisms account for thelack of a more substantial upregulation of PARIS. To avoid potentialdevelopmental compensation, exon 7 was deleted in 6-8 week oldconditional parkin KO mice in which exon 7 was flanked by loxP sites(parkin^(Flx/Flx)) by SN stereotactic injection of a GFP-fused Crerecombinase lentivirus (Lenti-GFPCre) and compared to control SNinjections of lentivirus expressing GFP (Lenti-GFP) in parkin^(Flx/Flx)mice (FIGS. 52 and 53). Four weeks after injection of the lentiviruses,Lenti-GFP and Lenti-GFPCre effectively transduced neurons in the SNincluding DA neurons (FIG. 52). Lenti-GFPCre led to almost a completeloss of parkin from the ventral midbrain of parkin^(Flx/Flx) micecompared to Lenti-GFP mice (FIGS. 53 and 54). Accompanying the loss ofparkin was greater than a two-fold upregulation of PARIS. Theupregulation of PARIS in the conditional parkin exon 7 KO model was notdue to a transcriptional effect since there was no alteration in PARISmRNA in the conditional parkin KO ventral midbrain versus WT ventralmidbrain (FIG. 42). Thus, the levels of PARIS were increased inconjunction with parkin inactivation and impairment ofubiquitin-mediated proteasomal degradation in sporadic PD, AR-PD and inan animal model of parkin inactivation.

Experiment 5 PARIS was a Transcriptional Repressor of PGC-1α

Proteins with KRAB domains can function as transcriptional repressors.GAL4-BD fused PARIS led to decreased luciferase activity from a5×GAL4-luciferase reporter construct, which was restored byco-expression of WT parkin, but not Q311X mutant parkin (FIG. 55). Toidentify the PARIS DNA binding consensus sequence, a chimeric proteincontaining the zinc finger domain (ZF) of PARIS (amino acids, 453-589)fused in frame to glutathione-S-tranferase (GST), GST-ZF-PARIS, was usedin a Cyclic Amplification and Selection of Targets (CAST) assay (FIG.56). Immobilized GST-ZF-PARIS was incubated with a pool ofoligonucleotides containing 26 random nucleotides. The final pool ofoligonucleotides remaining after four rounds of CASTing followed bythree rounds of electrophoretic mobility shift assays (EMSA) was cloned,sequenced and analyzed. Alignment of all the sequences using the programMACAW (National Center for Biotechnology Information) revealed aconsensus sequence with a core sequence composed of TATTTT (T/G) (FIG.56). 19 out of 24 sequences contained the core sequence and 3 out of 19sequence tags were identified as duplicates indicating this was theprimary DNA binding sequence for PARIS. The TATTTT (T/G) consensussequence was an insulin response sequence (IRS) designated thephosphoenolpyruvate carboxykinase (PEPCK)-like motif (PLM), which wasinvolved in the regulation of transcripts involved in energy metabolismand insulin responsiveness.

A NCBI survey of IRS/PLM responsive transcripts revealed that members ofthe PPARγ coactivator-1 (PGC-1) family of transcriptional co-activatorswere regulated by IRS sites. PGC-1α containd three IRS/PLM elementswithin its 5′-promoter region (FIG. 57). The activity of the 1 kb humanPGC-1α promoter was decreased approximately 40% in the presence ofPARIS, which was rescued by parkin overexpression (FIG. 57). Similarresults were observed with the 2 kb mouse PGC-1α promoter (FIG. 58). Thefamilial parkin mutant, Q311 X, had minimal effects on the PGC-1αpromoter (FIG. 57). PARIS repressed PGC-1α promoter activity by bindingto the IRS sites, since it failed to inhibit the reporter in which thethree IRS sites were deleted in the context of the PGC-1αpromoter-reporter construct (FIG. 57). Reporter constructs for PGC-1α(pGL3-h PGC-1α) were repressed by PARIS.

Individual IRS site mutants (IRS1-M, IRS2-M, IRS3-M) within the PGC-1αreporter that disrupted PARIS binding to the PGC-1α promoter asdetermined by EMSA (see FIG. 59) and a promoter construct, IRS123-M,containing all three mutations were evaluated in the promoter reporterassay (FIG. 60). IRS1-M and IRS3-M substantially reduced PGC-1α reporterpromoter activity, whereas IRS2-M increased the activity (FIG. 60).PARIS overexpression inhibited IRS1-M, IRS2-M, IRS3-M PGC-1α reporterpromoter activity suggesting that PARIS bound to and regulated all threesites (FIG. 60) as indicated from the EMSA assays (FIG. 59).

EMSA showed that PARIS bound to the PGC-1α IRS elements since GST-PARIS(full length) elicited a shift of [³²P]-labeled oligonucleotidescontaining the IRS1, IRS2, and IRS3 sequences of the PGC-1α promoter,whereas it failed to cause a shift of IRS1, IRS2 and IRS3 containing asingle base substitution within the IRS sequence (FIG. 59). Additionallyeight zinc finger mutants of PARIS were assessed for their ability torepress the PGC-1α reporter promoter construct (FIGS. 61 and 62) (seeTable 6). M1, M2, M8 PARIS mutants repressed PGC-1α reporter promoteractivity, similar to WT PARIS. M3, M4, M5, M6 PARIS mutants partiallyrepressed PGC-1α reporter promoter activity, whereas the M7 (C571A)PARIS mutant had no affect on PGC-1α reporter promoter activity. The M7GST-0571A-PARIS mutant had substantially reduced IRS-binding capacity asdetermined by EMSA (FIG. 63).

TABLE 6 Target genes Primers (5′-3′) M1 PARIS FACC TGC GCC ACG GCT GGG AAG AGC TTC (SEQ ID NO. 121) C458A RGAA GCT CTT CCC AGC CGT GGC GCA GGT (SEQ ID NO. 122) M2 PARIS FAGC CTG AGC GCG GCC CAG CGC AGC TGT (SEQ ID NO. 123) H471A RACA GCT GCG CTG GGC CGC GCT CAG GCT (SEQ ID NO. 124) M3 PARIS FGAG TGC GGC CGT GCC TTC ACG CGC CCC (SEQ ID NO. 125) C518A RGGG GCG CGT GAA GGC ACG GCC GCA CTC (SEQ ID NO. 126) M4 PARIS FCAC CTC ATC CGC GCT CGC ATG CTG CAC (SEQ ID NO. 127) H528A RGTG CAG CAT GCG AGC GCG GAT GAG GTG (SEQ ID NO. 128) M5 PARIS FTTC CCC TGC ACC GAG GCT GAG AAG CGC TTC (SEQ ID NO. 129) C543A RGAA GCG CTT CTC AGC CTC GGT GCA GGG GAA (SEQ ID NO. 130) M6 PARIS FCAC TAC CGA ACG GCC ACG GGC GTG CGG (SEQ ID NO. 131) H560A RCCG CAC GCC CGT GGC CGT TCG GTA GTG (SEQ ID NO. 132) M7 PARIS FACC TGC ACC GTC GCC GGC AAA AGC TTC (SEQ ID NO. 133) C571A RGAA GCT TTT GCC GGC GAC GGT GCA GGT (SEQ ID NO. 134) M8 PARIS FCAC CTC CGC AAG GCC CAG CGC AAC CAT (SEQ ID NO. 135) H584A RATG GTT GCG CTG GGC CTT GCG GAG GTG (SEQ ID NO. 136)

List of primers used for site-directed mutagenesis for PARIS used inFIGS. 61-66. Abbreviations: F, forward; R, reverse.

Chromatin immunoprecipitation (ChIP) indicated that PARIS bound to theendogenous PGC-1α promoter in SH-SY5Y cells (FIG. 67) and mouse brain(FIG. 68). PARIS also bound to the endogenous PGC-1α promoter in humanbrain (FIG. 40) and consistent with its upregulation in PD striatum (seeFIGS. 36 and 37) there was enhanced PARIS occupancy of endogenous PGC-1αin PD striatum compared to control (FIG. 41). Luciferase reporter assaywas performed in SH-SY5Y cells and ChIP assays in PD versus controlstriatum and SH-SY5Y cells with phosphoenolpyruvate carboxykinase(PEPCK) (FIG. 69) and glucose-6-phosphatase (G6Pase) (FIG. 70). Theluciferase reporter assay showed that overexpression of PARIS enhancedthe promoter activity of rat PEPCK, but not mouse G6Pase promoteractivity (FIG. 71). The ChIP assay demonstrated that PARIS bound to theendogenous promoter of human PEPCK and G6Pase in SH-SY5Y cells and incontrol and PD postmortem striatum (FIG. 72). In contrast to PGC-1α,there was not enhanced occupancy of the PEPCK and G6Pase promoter byPARIS. These data suggested that PARIS can bind to the promoter of PEPCKand G6Pase, but in contrast to PGC-1α it positively regulated PEPCK andit had no appreciable effect on G6Pase. Thus, the transcriptionalrepressive effects of PARIS were relatively specific to PGC-1α.

GFP-WT-PARIS overexpression led to approximately a 75% reduction inPGC-1α mRNA (FIG. 64) and approximately a 60% reduction in proteinlevels of PGC-1α (FIGS. 65 and 66), whereas the GFP-0571A-PARIS mutanthad no effect on PGC1-α protein or message levels (FIGS. 64-66).Lentiviral shRNA-parkin led to a two fold increase in the level of PARISfollowed by a 66% reduction of PGC-1α (FIGS. 73 and 74). To determinewhether the reduction in PGC-1α levels induced by the absence of parkinwas dependent on the presence of PARIS, a double knockdown experimentwas performed by lentiviral shRNA-parkin and/or shRNA-PARIS in SH-SY5Ycells. Knockdown of PARIS prevented the downregulation of PGC-1α levelsinduced by parkin knockdown (FIGS. 73 and 74). Knockdown of PARISresulted in a 3 fold increase in PGC-1α protein levels (FIGS. 73 and 74)and a 3.5 fold increase in PGC-1α mRNA (FIG. 75). Knockdown of PARIS inthe setting of parkin knockdown also prevented the down regulation ofPGC-1α mRNA (FIG. 75). These results taken together indicated that PARISwas a transcriptional repressor that negatively regulated the levels ofendogenous PGC-1α and that the downregulation of PGC-1α in the absenceof parkin was due to the upregulation of PARIS.

Experiment 6 Identification of NRF-1 as a potential in vivo PGC-1αtarget gene in PD

PGC-1α is a transcriptional coactivator that regulates a variety ofgenes. Real-time quantitative RT-PCR (qRT-PCR) was performed on avariety of PGC-1α target genes in PD SN and striatum to determine whichPGC-1α target genes are co-regulated by PARIS in PD.

The levels of PGC-1α co-regulated genes; which play important roles inmitochondrial function and oxidant metabolism, were measured. The PGC-1αco-regulated genes included nuclear respiratory factor-1 (NRF-1),copper/zinc superoxide dismutase (SOD1), manganese SOD (SOD2),glutathione peroxidase (GPx1), catalase (CAT), mitochondrial uncouplingproteins (UCP2 and UCP3), mitochondrial transcription factor A (Tfam)and the oxidative phosphorylation regulators, ATP5b, cytochrome C (CytC)and cytochrome C oxidase (COX II and IV) (FIGS. 38 and 39). In addition,the levels of other genes containing IRS/PLM in their promoter includingPEPCK, G6Pase, insulin-like-growth-factor binding protein 1 (IGFBP-1),tyrosine aminotransferase (TAT), and apolipoprotein C III (APOC3) weremonitored along with PGC-1α to assess whether PGC-1α is selectivelyaffected in PD (FIGS. 38 and 39). The levels of PARIS and parkin werealso assessed as controls.

It was found that PGC-1α and NRF-1 mRNA were downregulated in PD SN andstriatum compared to control SN and striatum (FIGS. 38 and 39). In PD SNATP5B was also significantly downregulated and CAT was significantlyupregulated (FIG. 38). All other PGC-1α dependent genes were notsignificantly altered (FIGS. 38 and 39). In addition there was nosignificant change in the levels of the IRS/PLM responsive transcriptsPEPCK, G6Pase and IGFBP-1 (FIGS. 38 and 39). TAT and APOC3 were notdetectable. No significant alteration in the mRNA level of PARIS andparkin was observed between PD and control SN and striatum (FIGS. 38 and39) indicating that the upregulation in PARIS protein levels (see FIGS.36 and 37) were most likely due to impairment of parkin E3 ubiquitinligase activity. Moreover, the absence of an alteration in the mRNAlevels of PARIS and parkin suggested that the changes in the mRNA levelsof PGC-1α and NRF-1 were specific and not due to the degenerativeprocess that occurred in PD.

As shown above (see FIGS. 36 and 37) PARIS protein was upregulatedalmost 3 fold in PD SN (FIGS. 43 and 44) and greater than two-fold in PDstriatum (FIGS. 45 and 46) compared to control SN and striatumrespectively. Accompanying the upregulation of PARIS was the downregulation of PGC-1α and NRF-1 in SN (FIGS. 43 and 44) and striatum(FIGS. 45 and 46). There was a trend towards redistribution of parkinfrom the soluble to insoluble fraction in SN (FIGS. 43 and 44) andparkin shifted from the soluble to insoluble fraction in PD striatum(FIGS. 45 and 46). There was a strong negative correlation between theprotein levels of PARIS and PGC-1α (R²=0.5195, p<0.05) and NRF-1(R²=0.8015, p<0.01) in the striatum and between PARIS and PGC-1α(R²=0.6955, p<0.05) and NRF-1 (R²=0.5979, p<0.05) in the SN and apositive correlation between PGC-1α and NRF-1 striatum (R²=0.6827,p<0.05) and SN(R²=0.6488, p<0.05) (FIG. 47). These results takentogether indicated that PARIS accumulated in the nigrostriatal pathwayin PD leading to the down regulation of PGC-1α and the PGC-1α dependentgene, NRF-1.

Experiment 7 Down Regulation of PGC-1α and NRF-1 in Conditional ParkinKO Mice Required PARIS

In adult conditional parkin KO mice (see FIGS. 52-54) four weeks afterLenti-GFPCre mediated parkin deletion there was a greater than two foldupregulation of PARIS (FIGS. 52-54, 76 and 77), comparable to that whichoccurred in sporadic PD SN (see FIGS. 36 and 37) and a concomitant downregulation of PGC-1α and NRF-1 (FIGS. 76 and 77). The alteration ofPGC-1α and NRF1 resulted from the reduction of their mRNA levels (FIG.78). Moreover, the mRNA levels of PGC-1α target genes in the ventralmidbrain of the Lenti-GFPCre-mediated conditional parkin KO wereanalyzed and showed a significant reduction of PGC-1α, SOD2, and NRF-1and no significant alteration in other sampled PGC-1α regulatedtranscripts (FIG. 42) similar to what occurred in sporadic PD brain. Inaddition, the levels of other genes containing IRS/PLM in their promoterincluding PEPCK, G6Pase, IGFBP-1, TAT, and APOC were monitored. Of thegenes containing IRS/PLM in their promoters, only PGC-1α wassignificantly down regulated (FIG. 42). The upregulation of PARIS andsubsequent downregulation of PGC-1α and NRF-1 occurred prior to the lossof DA neurons since there was no appreciable loss of DA neurons at 3months after the Lenti-GFPCre injection (see FIG. 79). Moreover, lasercapture microdissection (LCM) was performed prior to the loss of DAneurons to obtain mRNA from TH positive neurons transduced with GFP-Crefrom conditional parkin KO mice 4 weeks after the Lenti-GFPCre injectionto ascertain whether the reduction of PGC-1α was cell autonomous in DAneurons (FIGS. 80 and 81). There was a robust reduction of PGC-1α mRNAin TH-positive DA neurons of conditional parkin KO mice, whereas thelevels of PARIS mRNA were unchanged (FIGS. 80 and 81). PARIS was onlymodestly upregulated in germline parkin exon 7 KO mice (also see FIGS.49 and 50). PGC-1α and NRF-1 protein levels in germline parkin exon 7 KOmice were comparable to those of WT mice (FIGS. 82 and 83). Thus,germline deletion of parkin apparently led to compensatory changes thatprevented substantial alterations in the levels of PGC-1α and NRF-1.

The Cre-flox conditional parkin exon 7 KO model was developed byintroducing lentiviral shRNA-PARIS along with lenti-GFPCre intoparkin^(Flx/Flx) mice to address whether the changes in PGC-1α and NRF-1were due to PARIS (FIGS. 76-80). Co-administration of lentiviralshRNA-PARIS along with Lenti-GFPCre prevented the changes in PGC-1α andNRF-1 protein and mRNA as compared to control lentiviral shRNA-dsRedplus Lenti-GFPCre (FIGS. 76-80). These results taken together indicatedthat PARIS accumulated in the nigrostriatal pathway in PD and in modelsof parkin inactivation led to the down regulation of PGC-1α and thePGC-1α dependent gene, NRF-1. These changes in PGC-1α and NRF-1 due tothe loss of parkin were cell autonomous and preceded the loss of DAneurons and were due to the upregulation of PARIS, since knockdown ofPARIS prevented these changes. Moreover, since deletion of parkin fromadult mice led to similar events that occurred in sporadic PD, it waslikely that the absence and/or inactivation of parkin in PD led to PARISupregulation and impairment of PGC-1α signaling.

Experiment 8 Neurodegeneration in Conditional Parkin KO Mice RequiredPARIS

Conditional KO of parkin led to a significant reduction in tyrosinehydroxylase (TH) positive and NissI stained DA neurons 10 months afterstereotaxic injection of Lenti-GPFCre into the SN of 6-8 week oldparkin^(Flx/Flx) mice compared to parkin^(Flx/Flx) mice injected withcontrol Lenti-GFP (FIGS. 79 and 84). The loss of DA neurons wasprogressive; since there was no substantial loss of DA neurons after 3months (FIG. 79). PARIS was required for the loss of DA neurons inconditional parkin KO mice, since co-administration of lentiviralshRNA-PARIS along with Lenti-GFPCre significantly reduced the loss of DAneurons due to conditional KO of parkin (FIGS. 79 and 84). Takentogether these results indicated that conditional KO of parkin in adultmice led to degeneration of DA neurons and the upregulation of PARIS wasnecessary to contribute to the demise of DA neurons.

Experiment 9 Overexpression of PARIS Killed Dopamine Neurons In VivoRestoration by Parkin and PGC-1α

A PARIS overexpression model was developed in which AAV1-PARIS wasstereotactically injected into the SN of C57BI/6 mice and compared tomice injected with control AAV1-GFP virus. Stereotactic intranigralinjection of AAV1 effectively transduced the entire SN (FIG. 85). Onemonth after stereotactic injection of the viruses, AAV1 mediatedoverexpression of PARIS led to a greater than two-fold upregulation ofPARIS levels in the SN of mice (FIGS. 86 and 87) and it had no affect onparkin levels (FIGS. 86 and 87). Accompanying the increase in PARISlevels was a concomitant downregulation of PGC-1α and NRF-1 proteinlevels (FIGS. 86 and 87).

PARIS overexpression led to a greater than 40% reduction in TH positiveand NissI stained DA neurons positive neurons (FIGS. 88 and 89). Nosubstantial decrement in GABAergic neurons as assessed by GAD65/67immunoreactivity via immunohistochemistry was observed (FIG. 90) or viaimmunoblot (FIGS. 91 and 92) in AAV1-PARIS versus AAV1-GFP transducedSN, indicating that PARIS overexpression was selectively detrimental toDA neurons. Co-administration of AAV1-Parkin with AAV1-PARIS preventedthe loss of dopamine neurons induced by PARIS overexpression (FIGS. 88and 89). Lentiviral PGC-1α also prevented the loss of DA neurons inducedby PARIS overexpression (FIGS. 88 and 89). AAV1-mediated overexpressionof PARIS was confirmed by immunoblot analysis and reduced PGC-1α andNRF-1 levels by 52% and 60%, respectively. These reductions wererestored by co-overexpression of parkin or PGC-1α (FIGS. 93 and 94).These results indicated that PARIS overexpression was sufficient todownregulate PGC-1α, NRF-1 and selectively killed DA kills neuronsthrough a PGC-1α dependent mechanism.

Examples

Additional embodiments are described in the following paragraphs.

Paragraph 1. A method to diagnose Parkinson's disease by testing for anelevated level of PARIS.

Paragraph 2. A method to diagnose Parkinson's disease by testing for anelevated level of PARIS in urine.

Paragraph 3. A method to diagnose Parkinson's disease by testing for anelevated level of PARIS in blood.

Paragraph 4. A method to diagnose Parkinson's disease by testing for anelevated level of PARIS in cerebra-spinal fluid.

Paragraph 5. A method to diagnose Parkinson's disease by testing for anelevated level of PARIS in the brain.

Paragraph 6. A method to diagnose Parkinson's disease by testing for anelevated level of PARIS in a bodily fluid.

Paragraph 7. A method to diagnose Parkinson's disease by measuring aprotein level of PARIS.

Paragraph 8. A method to diagnose Parkinson's disease by measuring aprotein level of PARIS in urine.

Paragraph 9. A method to diagnose Parkinson's disease by measuring aprotein level of PARIS in blood.

Paragraph 10. A method to diagnose Parkinson's disease by measuring aprotein level of PARIS in cerebra-spinal fluid.

Paragraph 11. A method to diagnose Parkinson's disease by measuring aprotein level of PARIS in the brain.

Paragraph 12. A method to diagnose Parkinson's disease by measuring aprotein level of PARIS in a bodily fluid.

Paragraph 13. A method to diagnose Parkinson's disease by testing for anelevated level of a metabolite of PARIS.

Paragraph 14. A method to diagnose Parkinson's disease by testing for anelevated level of a metabolite of PARIS in urine.

Paragraph 15. A method to diagnose Parkinson's disease by testing for anelevated level of a metabolite of PARIS in blood.

Paragraph 16. A method to diagnose Parkinson's disease by testing for anelevated level of a metabolite of PARIS in cerebra-spinal fluid.

Paragraph 17. A method to diagnose Parkinson's disease by testing for anelevated level of a metabolite of PARIS in the brain.

Paragraph 18. A method to diagnose Parkinson's disease by testing for anelevated level of a metabolite of PARIS in a bodily fluid.

Paragraph 19. A method to diagnose Parkinson's disease by measuring aprotein level of a metabolite of PARIS.

Paragraph 20. A method to diagnose Parkinson's disease by measuring aprotein level of a metabolite of PARIS in urine.

Paragraph 21. A method to diagnose Parkinson's disease by measuring aprotein level of a metabolite of PARIS in blood.

Paragraph 22. A method to diagnose Parkinson's disease by measuring aprotein level of a metabolite of PARIS in cerebra-spinal fluid.

Paragraph 23. A method to diagnose Parkinson's disease by measuring aprotein level of a metabolite of PARIS in the brain.

Paragraph 24. A method to diagnose Parkinson's disease by measuring aprotein level of a metabolite of PARIS in a bodily fluid.

Paragraph 25. A method to diagnose Parkinson's disease by measuringlevel of an mRNA coding for PARIS.

Paragraph 26. A method to diagnose Parkinson's disease by measuringlevel of an mRNA coding for PARIS in urine.

Paragraph 27. A method to diagnose Parkinson's disease by measuringlevel of an mRNA coding for PARIS in blood.

Paragraph 28. A method to diagnose Parkinson's disease by measuringlevel of an mRNA coding for PARIS in cerebra-spinal fluid.

Paragraph 29. A method to diagnose Parkinson's disease by measuringlevel of an mRNA coding for PARIS in blood the brain.

Paragraph 30. A method to diagnose Parkinson's disease by measuringlevel of a mRNA coding for PARIS in a bodily fluid.

Paragraph 31. A method to diagnose Parkinson's disease by testing for areduced level of PGC-1α.

Paragraph 32. A method to diagnose Parkinson's disease by testing for areduced level of PGC-1α in urine.

Paragraph 33. A method to diagnose Parkinson's disease by testing for areduced level of PGC-1α in blood.

Paragraph 34. A method to diagnose Parkinson's disease by testing for areduced level of PGC-1α in cerebra-spinal fluid.

Paragraph 35. A method to diagnose Parkinson's disease by testing for areduced level of PGC-1α in the brain.

Paragraph 36. A method to diagnose Parkinson's disease by testing for areduced level of PGC-1α in a bodily fluid.

Paragraph 37. A method to diagnose Parkinson's disease by measuringprotein level of PGC-1α.

Paragraph 38. A method to diagnose Parkinson's disease by measuringprotein level of PGC-1α in urine.

Paragraph 39. A method to diagnose Parkinson's disease by measuringprotein level of PGC-1α in blood.

Paragraph 40. A method to diagnose Parkinson's disease by measuringprotein level of PGC-1α in cerebra-spinal fluid.

Paragraph 41. A method to diagnose Parkinson's disease by measuringprotein level of PGC-1α in the brain.

Paragraph 42. A method to diagnose Parkinson's disease by measuringprotein level of PGC-1α in a bodily fluid.

Paragraph 43. A method to diagnose Parkinson's disease by measuringlevel of an mRNA coding for PGC-1α.

Paragraph 44. A method to diagnose Parkinson's disease by measuringlevel of an mRNA coding for PGC-1α in urine.

Paragraph 45. A method to diagnose Parkinson's disease by measuringlevel of an mRNA coding for PGC-1α in blood.

Paragraph 46. A method to diagnose Parkinson's disease by measuringlevel of an mRNA coding for PGC-1α in cerebra-spinal fluid.

Paragraph 47. A method to diagnose Parkinson's disease by measuringlevel of an mRNA coding for PGC-1α in the brain.

Paragraph 48. A method to diagnose Parkinson's disease by measuringlevel of an mRNA coding for PGC-1α in a bodily fluid.

Paragraph 49. A method to identify small molecular compound that can beused to treat Parkinson's disease.

Paragraph 50. A method to identify small molecular compound that can beused to treat Parkinson's disease related disorders.

Paragraph 51. A reporter construct for PGC-1α (pGL3-h PGC-1α) that isrepressed by PARIS.

Paragraph 52. A SK-SHSY cell line to stably express PARIS and pGL3-hPGC-1α, and GL3-h PGC-1α alone, wherein PARIS and pGL3-h PGC-1α are usedto screen for PARIS inhibitors.

Paragraph 53. A method to identify small molecule compounds of PARISthat leave unaffected other regulatory signaling of PGC-1α that is PARISindependent.

Paragraph 54. An in vitro model of PARIS overexpression that can be usedto validate a PARIS inhibitor.

Paragraph 55. An in vitro model of PARIS overexpression that can be usedto optimize a PARIS inhibitor.

Paragraph 56. An in vivo model of PARIS overexpression that can be usedto validate a PARIS inhibitor.

Paragraph 57. An in vivo model of PARIS overexpression that can be usedto optimize a PARIS inhibitor.

Paragraph 58. An in vitro model of Parkin inactivation that can be usedto validate a PARIS inhibitor.

Paragraph 59. An in vitro model of Parkin inactivation that can be usedto optimize a PARIS inhibitor.

Paragraph 60. An in vivo model of Parkin inactivation that can be usedto validate a PARIS inhibitor.

Paragraph 61. An in vivo model of Parkin inactivation that can be usedto optimize a PARIS inhibitor.

Paragraph 62. A method to select inhibitors of PARIS by disrupting afunction of PARIS.

Paragraph 63. A method to develop biologic assays to confirm and/orcharacterize a PARIS inhibitor.

Paragraph 64. A method to determine the effect of an inhibitor of PARISon neuronal viability in model of Parkinson's disease.

Paragraph 65. An isolated nucleotide of SEQ ID NO. 27.

Paragraph 66. An isolated nucleotide of SEQ ID NO. 28.

Paragraph 67. A method of treating Parkinson's disease by administeringa shRNA inhibitor.

Paragraph 68. A method of treating Parkinson's disease by administeringan anti-sense microRNA inhibitor.

Paragraph 69. A method of treating Parkinson's disease related disordersby administering a shRNA inhibitor.

Paragraph 70. A method of treating Parkinson's disease related disordersby administering an anti-sense microRNA inhibitor.

Paragraph 71. A method of treating neurodegenerative and relatedneurologic diseases, such as, Alexander's disease, Alper's disease,Alzheimer's disease, amyotrophic lateral sclerosis, ataxiatelangiectasia, Batten disease, bovine spongiform encephalopathy,Canavan disease, Cockayne syndrome, corticobasal degeneration,Creutzfeldt-Jakob disease, Huntington's disease, HIV associateddementia, Kennedy's disease, Krabbe's disease, lewy body dementia,Machado-Joseph disease, multiple sclerosis, multiple system atrophy,narcolepsy, neuroborreliosis, Parkinson's disease, Pelizaeus-MerzbacherDisease, Pick's disease, primary lateral sclerosis, prion diseases,Refsum's disease, Sandhoff's disease, Schilder's disease, subacutecombined degeneration of spinal cord secondary to pernicious anemia,schizophrenia, spinocerebellar ataxia, spinal muscular atrophy,Steele-Richardson-Olszewski disease, and tabes dorsalis; byadministering inhibitors of PARIS.

Paragraph 72. A method of treating a metabolic disorder, such as,diabetes mellitus, dyslipidemia, and obesity, by administering aninhibitor of PARIS.

Paragraph 73. A method of treating a circulatory disorder, such as,atherosclerosis, cardiovascular disease, and cardiac ischemia, byadministering an inhibitor of PARIS.

Paragraph 74. A method of treating an inflammatory condition such asinflammatory bowel diseases, colitis and psoriasis; by administering aninhibitor of PARIS.

Paragraph 75. A method of treating a cancer by administering aninhibitor of PARIS.

Paragraph 76. A method of treating a kidney disease includingglomerulonephritis, glomerulosclerosis and diabetic nephropathy byadministering an inhibitor of PARIS.

Paragraph 77. A method of treating a mitochondrial disorder byadministering an inhibitor of PARIS.

Paragraph 78. A method of treating a muscle disorder including musculardystrophies by administering an inhibitor of PARIS.

Paragraph 79. A method of treating a disorder of circadian rhythms andsleep by administering an inhibitor of PARIS.

Paragraph 80. An isolated polypeptide comprising of any of SEQ ID NO: 1to SEQ ID NO: 4.

Paragraph 81. An isolated polypeptide consisting of any of SEQ ID NO: 1to SEQ ID NO: 4.

Paragraph 82. An isolated polypeptide comprising a peptide sequence ofany of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 83. An isolated polypeptide comprising the peptide sequence ofany of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 84. An isolated polypeptide comprising a polypeptide coded bythe nucleotide sequence of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 85. An isolated polynucleotide comprising of any of SEQ ID NO:5 to SEQ ID NO: 136.

Paragraph 86. An isolated polynucleotide consisting of any of SEQ ID NO:5 to SEQ ID NO: 136.

Paragraph 87. An isolated polynucleotide comprising a nucleotidesequence of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 88. An isolated polynucleotide comprising the nucleotidesequence of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 89. An isolated polynucleotide comprising a polynucleotideencoding the amino acid sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 90. An isolated polynucleotide comprising a polynucleotidewhich hybridizes under stringent conditions to any of SEQ ID NO: 5 toSEQ ID NO: 136.

Paragraph 91. A composition comprising an isolated polypeptidecomprising of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 92. A composition comprising an isolated polypeptideconsisting of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 93. A composition comprising an isolated polypeptidecomprising a peptide sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 94. A composition comprising an isolated polypeptidecomprising the peptide sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 95. A composition comprising an isolated polypeptidecomprising a polypeptide coded by the nucleotide sequence of any of SEQID NO: 5 to SEQ ID NO: 136.

Paragraph 96. A composition comprising an isolated polynucleotidecomprising of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 97. A composition comprising an isolated polynucleotideconsisting of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 98. A composition comprising an isolated polynucleotidecomprising a nucleotide sequence of any of SEQ ID NO: 5 to SEQ ID NO:136.

Paragraph 99. A composition comprising an isolated polynucleotidecomprising the nucleotide sequence of any of SEQ ID NO: 5 to SEQ ID NO:136.

Paragraph 100. A composition comprising an isolated polynucleotidecomprising a polynucleotide encoding the amino acid sequence of any ofSEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 101. A composition comprising an isolated polynucleotidecomprising a polynucleotide which hybridizes under stringent conditionsto any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 102. A composition consisting of an isolated polypeptidecomprising of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 103. A composition consisting of an isolated polypeptideconsisting of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 104. A composition consisting of an isolated polypeptidecomprising a peptide sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 105. A composition consisting of an isolated polypeptidecomprising the peptide sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 106. A composition consisting of an isolated polypeptidecomprising a polypeptide coded by the nucleotide sequence of any of SEQID NO: 5 to SEQ ID NO: 136.

Paragraph 107. A composition consisting of an isolated polynucleotidecomprising of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 108. A composition consisting of an isolated polynucleotideconsisting of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 109. A composition consisting of an isolated polynucleotidecomprising a nucleotide sequence of any of SEQ ID NO: 5 to SEQ ID NO:136.

Paragraph 110. A composition consisting of an isolated polynucleotidecomprising the nucleotide sequence of any of SEQ ID NO: 5 to SEQ ID NO:136.

Paragraph 111. A composition consisting of an isolated polynucleotidecomprising a polynucleotide encoding the amino acid sequence of any ofSEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 112. A composition consisting of an isolated polynucleotidecomprising a polynucleotide which hybridizes under stringent conditionsto any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 113. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of an isolatedpolypeptide comprising of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 114. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of an isolatedpolypeptide consisting of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 115. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of an isolatedpolypeptide comprising a peptide sequence of any of SEQ ID NO: 1 to SEQID NO: 4.

Paragraph 116. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of an isolatedpolypeptide comprising the peptide sequence of any of SEQ ID NO: 1 toSEQ ID NO: 4.

Paragraph 117. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of an isolatedpolypeptide comprising a polypeptide coded by the nucleotide sequence ofany of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 118. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of an isolatedpolynucleotide comprising of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 119. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of an isolatedpolynucleotide consisting of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 120. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of an isolatedpolynucleotide comprising a nucleotide sequence of any of SEQ ID NO: 5to SEQ ID NO: 136.

Paragraph 121. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of an isolatedpolynucleotide comprising the nucleotide sequence of any of SEQ ID NO: 5to SEQ ID NO: 136.

Paragraph 122. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of an isolatedpolynucleotide comprising a polynucleotide encoding the amino acidsequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 123. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of an isolatedpolynucleotide comprising a polynucleotide which hybridizes understringent conditions to any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 124. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositioncomprising an isolated polypeptide comprising of any of SEQ ID NO: 1 toSEQ ID NO: 4.

Paragraph 125. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositioncomprising an isolated polypeptide consisting of any of SEQ ID NO: 1 toSEQ ID NO: 4.

Paragraph 126. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositioncomprising an isolated polypeptide comprising a peptide sequence of anyof SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 127. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositioncomprising an isolated polypeptide comprising the peptide sequence ofany of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 128. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositioncomprising an isolated polypeptide comprising a polypeptide coded by thenucleotide sequence of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 129. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositioncomprising an isolated polynucleotide comprising of any of SEQ ID NO: 5to SEQ ID NO: 136.

Paragraph 130. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositioncomprising an isolated polynucleotide consisting of any of SEQ ID NO: 5to SEQ ID NO: 136.

Paragraph 131. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositioncomprising an isolated polynucleotide comprising a nucleotide sequenceof any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 132. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositioncomprising an isolated polynucleotide comprising the nucleotide sequenceof any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 133. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositioncomprising an isolated polynucleotide comprising a polynucleotideencoding the amino acid sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 134. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositioncomprising an isolated polynucleotide comprising a polynucleotide whichhybridizes under stringent conditions to any of SEQ ID NO: 5 to SEQ IDNO: 136.

Paragraph 135. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositionconsisting of an isolated polypeptide comprising of any of SEQ ID NO: 1to SEQ ID NO: 4.

Paragraph 136. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositionconsisting of an isolated polypeptide consisting of any of SEQ ID NO: 1to SEQ ID NO: 4.

Paragraph 137. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositionconsisting of an isolated polypeptide comprising a peptide sequence ofany of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 138. A composition consisting of an isolated polypeptidecomprising the peptide sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 139. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositionconsisting of an isolated polypeptide comprising a polypeptide coded bythe nucleotide sequence of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 140. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositionconsisting of an isolated polynucleotide comprising of any of SEQ ID NO:5 to SEQ ID NO: 136.

Paragraph 141. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositionconsisting of an isolated polynucleotide consisting of any of SEQ ID NO:5 to SEQ ID NO: 136.

Paragraph 142. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositionconsisting of an isolated polynucleotide comprising a nucleotidesequence of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 143. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositionconsisting of an isolated polynucleotide comprising the nucleotidesequence of any of SEQ ID NO: 5 to SEQ ID NO: 136.

Paragraph 144. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositionconsisting of an isolated polynucleotide comprising a polynucleotideencoding the amino acid sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4.

Paragraph 145. A method of treatment for Parkinson's disease, comprisingadministering a pharmaceutically effective amount of a compositionconsisting of an isolated polynucleotide comprising a polynucleotidewhich hybridizes under stringent conditions to any of SEQ ID NO: 5 toSEQ ID NO: 136.

Paragraph 145. A kit comprising an isolated polynucleotide selected fromthe group consisting of sequences SEQ ID NO: 5 to SEQ ID NO: 136 andinstructions on their use.

Paragraph 145. A kit comprising an isolated polypeptide selected fromthe group consisting of sequences SEQ ID NO: 1 to SEQ ID NO: 4 andinstructions on their use.

Paragraph 145. A kit comprising the composition of any of Paragraphs 91to 112 and instructions on their use.

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The invention claimed is:
 1. A method to diagnose Parkinson's disease ina subject comprising the steps of: extracting nucleic acid from a testsample obtained from the subject; measuring an amount of a component ina test sample obtained from the subject, wherein the component isselected from the group consisting of Parkin Interacting Substrate(PARIS), a metabolite of Parkin interacting Substrate (PARIS), an mRNAcoding for Parkin Interacting Substrate (PARIS), peroxisomeproliferator-activated receptor gamma coactivator 1-α, (PGC-1α), and anmRNA coding for PGC-1α, using at least one primer pair selected from thegroup consisting of: (a) a first primer pair consisting of a reverseprimer consisting of SEQ ID NO: 120 and a forward primer consisting ofSEQ ID NO: 119; (b) a second primer pair consisting of a reverse primerconsisting of SEQ ID NO: 32 and a forward primer consisting of SEQ IDNO: 31; and (c) a third primer pair consisting of a reverse primerconsisting of SEQ ID NO: 76 and a forward primer consisting of SEQ IDNO: 75; determining the expression of the component; and comparing theamount of the component against a baseline expression value to determinewhether the levels of PARIS, metabolite of PARIS, an mRNA coding forPARIS are elevated or to determine whether the levels of PGC-1α, or anmRNA coding for PGC-1α are reduced as compared to levels of a healthysubject.
 2. The method of claim 1 wherein the test sample is a bodyfluid.
 3. The method of claim 2 wherein the body fluid is urine, blood,or cerebra-spinal fluid.